Methods and compositions for labeling nucleic acids

ABSTRACT

The present invention relates to methods for the labeling of nucleic acid polymers in vitro and in vivo. In particular, the methods include a [3+2] cycloaddition between a nucleotide analogue incorporated into a nucleic acid polymer and a reagent attached to a label. Such methods do not require fixation and denaturation and therefore can be applied to the labeling of nucleic acid polymers in living cells and in organisms. Also provided are methods for measuring cellular proliferation. In these methods, the amount of label incorporated into the DNA is measured as an indication of cellular proliferation. The methods of the invention can be used in a wide variety of applications including clinical diagnosis of diseases and disorders in which cellular proliferation is involved, toxicity assays, and as a tool for the study of chromosomes&#39; ultrastructures.

RELATED APPLICATIONS

The present invention claims priority from Provisional Application No.60/730,745, filed on Oct. 27, 2005 and entitled “Methods andCompositions for Labeling Nucleic Acids”. The Provisional Application isincorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

The work described herein was funded by the National Institutes ofHealth (Grant No. GM039565).

BACKGROUND OF THE INVENTION

Cell division and cell death play central roles in the properdevelopment of multi-cellular organisms and in the homeostaticmaintenance of tissues. Loss or reduction of cell proliferativecapability and dysregulation of cell death are among the most importantphenomena that characterize the aging process (D. Monti et al., Am. J.Clin. Nutr., 1992, 55 (6 Suppl): 1208S-1214S; H. R. Warner et al., J.Am. Geriatr. Soc., 1997, 45: 1140-1146; L. Ginaldi et al., Immunol.Res., 2000, 21: 31-38). Disruption of normal control of cellproliferation and cell death also underlies many pathological conditionsincluding cancer; infectious diseases such as acquired immunodeficiencysyndrome (J. C. Bentin et al., J. Clin. Immunol., 1989, 9: 159-168; R.A. Gruters et al., Eur. J. Immunol., 1990, 20: 1039-1044; L. Meyaard etal., Science, 1992, 257: 217-119; A. Cayota et al., Clin. Exp. Immunol.,1992, 88: 478-483); vascular disorders such as atherosclerosis andhypertension (S. M. Schwartz et al., Circ. Res., 1986, 58: 427-444; A.Rivard and V. Andres, Histol. Histopathol., 2000, 15: 557-571); andneurodegenerative diseases such as Alzheimer's disease (Z. Nagy, J.Neural Transm. Suppl., 1999, 57: 233-245; A. K. Raina et al., Prog. CellCycle Res., 2000, 4: 235-242; I. Vincent et al., Prog. Cell Cycle Res.,2003, 5: 31-41).

The most characteristic biochemical feature of cell division is DNAsynthesis, which occurs essentially only during the S phase of the cellcycle (S. Sawada et al., Mutat. Res., 1995, 344: 109-116). Accordingly,the most commonly used methods for the study of cell cycle, DNAsynthesis and cell proliferation rely on incorporation of labeledbiosynthetic precursors into the newly synthesized DNA of proliferatingcells (M. Bick and R. L. Davidson, Proc. Natl. Acad. Sci. USA, 1974, 71:2082-2086; H. G. Gratzner, Science, 1982, 218: 474-475; F. M. Waldman etal., Mod. Pathol., 1991, 4: 718-722). In these methods, labeled DNAprecursors (e.g., [³H]-thymidine or 5-bromo-2′-deoxyuridine (BrdU)) areadded to cells during replication, and their incorporation into genomicDNA is quantified following incubation and sample preparation.Incorporated [³H]-thymidine is generally detected by autoradiography.Detection of incorporated BrdU is performed immunologically after sampledenaturation to allow access of monoclonal antibodies, and the resultingBrdU-labeled cells are then analyzed by flow cytometry or microscopy. Tostudy cellular proliferation of specific tissues, animals areadministered (e.g., injected) labeled DNA precursors, sacrificed, andthe tissues are removed and fixed for microscopic analysis.

Although [³H]-thymidine and BrdU incorporation labeling methods haveproven valuable for studying cell cycle kinetics, DNA synthesis andsister chromatid exchange, as well as for assessing cell proliferationof normal or pathological cells or tissues under different conditions,these methods exhibit several limitations. The most notable disadvantageof [³H]-thymidine incorporation results from the complications and risksof using radioactivity. In addition, autoradiography is labor-intensiveand time-consuming. Furthermore, because both methods are sampledestructive, quantification can be performed at only one predeterminedtime point, and continuous monitoring of a single sample is notpossible. Additionally, in contrast to [³H]-thymidine autoradiography,BrdU immunohistochemistry is not stoichiometric (R. S, Nowakowski etal., J. Neurocytol., 1989, 18: 311-318; R. S, Nowakowski and N. L.Hayes, Science, 2000, 288: 771). Thus, the intensity or extent oflabeling is highly dependent on the conditions used for detection anddoes not necessarily reflect the magnitude of DNA replication. For thisreason, BrdU labeling as a measure of cell division is especiallyvulnerable to misinterpretation (P. Rakic, Nature Rev. Neurosci., 2002,3: 56-71).

More recently, a stable isotope-mass spectrometric technique has beendeveloped that resolves some of the problems associated with the[³H]-thymidine and BrdU incorporation methods (D. C. Macallan et al.,Proc. Natl. Acad. Sci. USA, 1998, 95: 708-713; M. K. Hellerstein,Immunol. Today, 1999, 20: 438-441; M. Hellerstein et al., Nature Med.,1999, 5: 83-89; J. M. McCune et al., J. Clin. Invest., 2000, 105: R1-8;H. Mohri et al., J. Exp. Med., 2001, 94: 1277-1288; R. M. Ribeiro etal., Proc. Natl. Acad. Sci. USA, 2002, 99: 15572-15577, R. M. Ribeiro etal., Bull. Math. Biol., 2002, 64: 385-405). In this technique, thedeoxyribose moiety of nucleotides in replicating DNA is labeledendogenously, through the de novo nucleotide synthesis pathway by usingstable isotope ²H- or ¹³C-labeled glucose. The isotopic enrichment ofthe DNA is then detected and quantified by gas chromatographic/massspectrometric (GC/MS) analysis after isolation, denaturation andhydrolysis of genomic DNA and TMS (trimethylsyl) derivatization of theresulting deoxyribonucleosides. Although this method has severaladvantages including being safe for use in humans, it has disadvantagesincluding that it involves a lengthy and destructive processing of thesample prior to detection.

Clearly, improved nucleic acid labeling techniques are still needed forthe study of cell cycle kinetics, DNA synthesis and cellularproliferation in vitro and in vivo. In particular, the development oftechniques that are simple, rapid, and sensitive and that do not requireextensive sample preparation and/or do not result in sample destructionremains highly desirable.

SUMMARY OF THE INVENTION

The present invention is directed to improved systems and strategies forstudying cell division. More specifically, the present inventionprovides methods and compositions useful for labeling nucleic acidmolecules and for measuring cellular proliferation in vitro and in vivo.In general, the inventive methods include a chemical reaction between anucleotide analogue incorporated into a nucleic acid polymer and areagent comprising a label, wherein the nucleotide analogue comprises afirst reactive group and the reagent comprises a second reactive groupsuch the reaction between the first and second reactive groups resultsin labeling of the nucleic acid polymer. Such methods do not requireextensive processing of the labeled sample. In particular, manyinventive methods do not require denaturation of the sample. Inparticular, methods are provided that include a [3+2] cycloadditionbetween a nucleotide analogue incorporated into a nucleic acid polymerand a reagent attached to a label.

More specifically, in one aspect, the present invention provides methodsfor labeling a nucleic acid polymer, comprising steps of: providing anucleic acid polymer containing at least one nucleotide analogue thatcomprises a first reactive unsaturated group; and contacting the nucleicacid polymer with a reagent comprising a second reactive unsaturatedgroup attached to a label, such that a [3+2] cycloaddition occursbetween the first and second unsaturated groups.

In certain embodiments, the first reactive unsaturated group comprises a1,3-dipole and the second reactive unsaturated group comprises adipolarophile. In other embodiments, the first reactive unsaturatedgroup comprises a dipolarophile and the second reactive unsaturatedgroup comprises a 1,3-dipole. In some embodiments, the 1,3-dipole maycomprise an azide group and the dipolarophile may comprise an ethynylgroup.

In certain embodiments, the label is directly detectable. For example,the label comprises a fluorescent agent. In other embodiments, the labelis indirectly detectable. For example, the label comprises a hapten.

The nucleic acid polymer to be labeled may be inside a cell, in a tissueor an organism.

In certain embodiments, the at least one nucleotide analogue isincorporated into the nucleic acid polymer during DNA replication or DNAtranscription.

In certain embodiments, the step of contacting the nucleic acid polymerwith a reagent is performed under aqueous conditions. The contacting maybe performed in the presence of Cu(I). Alternatively, the contacting maybe performed in the absence of Cu(I) with a reagent that furthercomprises a Cu chelating moiety.

In another aspect, the present invention provides methods for duallylabeling a nucleic acid polymer. The inventive methods comprise stepsof: providing a nucleic acid polymer containing at least one firstnucleotide analogue that comprises a first reactive unsaturated groupand at least one second nucleotide analogue that comprises a secondreactive unsaturated group; contacting the nucleic acid polymer with afirst reagent comprising a third reactive unsaturated group attached toa first label, such that a [3+2] cycloaddition occurs between the firstand third unsaturated groups; and contacting the nucleic acid polymerwith a second reagent comprising a fourth reactive unsaturated groupattached to a second label, such that a [3+2] cycloaddition occursbetween the second and fourth unsaturated groups.

In certain embodiments, the first reactive unsaturated group comprises afirst 1,3-dipole and the third reactive unsaturated group comprises afirst dipolarophile; the second reactive unsaturated group comprises asecond dipolarophile and the fourth reactive unsaturated group comprisesa second 1,3-dipole.

In other embodiments, the first reactive unsaturated group comprises afirst dipolarophile and the third reactive unsaturated group comprises afirst 1,3-dipole; the second reactive unsaturated group comprises asecond 1,3-dipole and the fourth reactive unsaturated group comprises asecond dipolarophile. As described above, a 1,3-dipole may comprise anazide group, and a dipoloraphile may comprise an ethynyl group.

In certain embodiments, the first and second labels are directlydetectable. In some such embodiments, the first label comprises a firstfluorescent agent, the second label comprises a second fluorescentagent, and the first and second fluorescent agents produce a dual-colorfluorescence upon excitation.

In other embodiments, the first and second labels are indirectlydetectable. For example, the first label comprises a first hapten andthe second label comprises a second hapten.

As mentioned above, the nucleic acid polymer to be dually labeled may beinside a cell, in a tissue or an organism; and the first and secondnucleotide analogues may be incorporated into the nucleic acid polymerduring DNA replication or DNA transcription.

In these inventive methods, the steps of contacting may be performedsimultaneously or sequentially. Preferably, the steps of contacting areperformed under aqueous conditions.

In another aspect, the present invention provides methods fordifferentially labeling nucleic acid polymers. These inventive methodscomprise steps of: providing a first nucleic acid polymer containing atleast one first nucleotide analogue that comprises a first reactiveunsaturated group; providing a second nucleic acid polymer containing atleast one second nucleotide analogue that comprises a second reactiveunsaturated group; contacting the first nucleic acid polymer with afirst reagent comprising a third reactive unsaturated group attached toa first label, such that a [3+2] cycloaddition occurs between the firstand third unsaturated groups; and contacting the second nucleic acidpolymer with a second reagent comprising a fourth reactive unsaturatedgroup attached to a second label, such that a [3+2] cycloaddition occursbetween the second and fourth unsaturated groups.

The first and second reactive unsaturated groups and first and secondlabels may be as described above. As described above, the steps ofcontacting may be performed simultaneously or sequentially.

In certain embodiments, the first nucleic acid polymer is inside a firstcell and the second polymer is inside a second cell. In otherembodiments, the first nucleic acid polymer is in a first tissue and thesecond nucleic acid polymer is in a second tissue. In still otherembodiments, the first nucleic acid polymer is in a first organism andthe second polymer is in a second organism.

In another aspect, the present invention provides nucleic acid polymerscomprising at least one nucleotide analogue attached to a label.Preferably, the nucleic acid polymers are prepared by one of thelabeling methods disclosed herein.

In certain embodiments, the nucleotide analogue incorporated into aninventive nucleic acid polymer comprises a cycloadduct, such as acycloadduct resulting from a [3+2] cycloaddition between an ethynylgroup and an azide group.

In certain embodiments, the label is covalently attached to thenucleotide analogue.

In certain embodiments, the label is directly detectable. For example,the label is a fluorescent agent. In other embodiments, the label isindirectly detectable. For example, the label comprises a hapten.

The present invention also provides dually labeled nucleic acidpolymers. More specifically, the present invention provides nucleic acidpolymers comprising at least one first nucleotide analogue attached to afirst label and at least one second nucleotide analogue attached to asecond label. Preferably, such nucleic acid polymers are prepared by thedual labeling methods described herein.

In certain embodiments, the at least one first nucleotide analoguecomprises a first cycloadduct resulting from a [3+2] cycloadditionbetween a first ethynyl group and a first azide group and the at leastone second nucleotide analogue comprises a second cycloadduct resultingfrom a [3+2] cycloaddition between a second ethynyl group and a secondazide group.

In certain embodiments, the first label comprises a first fluorescentagent, the second label comprises a second fluorescent agent, and thefirst and second fluorescent agent produce a dual fluorescence uponexcitation.

In another aspect, the present invention provides cells comprising oneor more inventive labeled or dually labeled nucleic acid polymers.

In still another aspect, the present invention provides kits forlabeling a nucleic acid polymer comprising: at least one nucleosideanalogue that comprises a first reactive unsaturated group; and areagent comprising a second reactive unsaturated group attached to alabel.

In certain embodiments, the first reactive unsaturated group comprises a1,3-dipole, the second reactive unsaturated group comprises adipolarophile and the first and second reactive unsaturated groups canreact via [3+2] cycloaddition. In other embodiments, the first reactiveunsaturated group comprises a dipolarophile, the second reactiveunsaturated group comprises a 1,3-dipole, and the first and secondreactive unsaturated groups can react via [3+2] cycloaddition.

The present invention also provides kits for dually labeling a nucleicacid polymer, comprising: at least one first nucleoside analogue thatcomprises a first reactive unsaturated group; at least one secondnucleoside analogue that comprises a second reactive unsaturated group;a first reagent comprising a third reactive unsaturated group attachedto a first label; and a second reagent comprising a fourth reactiveunsaturated group attached to a second label.

In certain embodiments, the first reactive unsaturated group comprises afirst 1,3-dipole, the third reactive unsaturated group comprises a firstdipolarophile, and the first and third reactive unsaturated groups canreact via [3+2] cycloaddition; the second reactive unsaturated groupcomprises a dipolarophile, the fourth reactive unsaturated groupcomprises a second 1,3-dipole, and the second and fourth reactiveunsaturated groups can react via [3+2] cycloaddition.

In other embodiments, the first reactive unsaturated group comprises afirst dipolarophile, the third reactive unsaturated group comprises afirst 1,3-dipole, and the first and third reactive unsaturated groupscan react via [3+2] cycloaddition; the second reactive unsaturated groupcomprises a dipolarophile, the fourth reactive unsaturated groupcomprises a second 1,3-dipole, and the second and fourth reactiveunsaturated groups can react via [3+2] cycloaddition. The inventive kitsmay further comprise aqueous medium and/or Cu(I).

In still another aspect, the present invention provides methods formeasuring cellular proliferation in a cell or in an organism.

Certain inventive methods comprise steps of: contacting a cell with aneffective amount of a nucleoside analogue that comprises a firstreactive unsaturated group such that the nucleoside analogue isincorporated into DNA of the cell; contacting the cell with a reagentcomprising a second reactive unsaturated group attached to a label, suchthat a [3+2] cycloaddition occurs between the first and second reactiveunsaturated groups; and determining an amount of label incorporated intothe DNA to measure cellular proliferation. The cell may be in amulti-well assay plate.

Other inventive methods comprise steps of: administering to an organisman effective amount of a nucleoside analogue that comprises a firstreactive unsaturated group such that the nucleoside analogue isincorporated into DNA of cells of the organism; contacting at least onecell of the organism with a reagent comprising a second reactiveunsaturated group attached to a label, such that a [3+2] cycloadditionoccurs between the first and second reactive unsaturated groups; anddetermining an amount of label incorporated into the DNA to measurecellular proliferation in the organism.

The first and second reactive unsaturated groups; labels and contactingsteps in these methods may be as described above.

In yet another aspect, the present invention provides methods foridentifying an agent that perturbs cellular proliferation in a cell orin an organism.

Certain inventive methods comprise steps of: (a) contacting a cell witha test agent; (b) contacting the cell with an effective amount of anucleoside analogue that comprises a first reactive unsaturated groupsuch that the nucleoside analogue is incorporated into DNA of the cell;(c) contacting the cell with a reagent comprising a second reactiveunsaturated group attached to a label, such that a [3+2] cycloadditionoccurs between the first and second reactive unsaturated groups; (d)determining an amount of label incorporated into the DNA, wherein theamount of label indicates the extent of cellular proliferation; and (e)identifying the test agent as an agent that perturbs cellularproliferation if the amount of label measured in step (d) is less thanor greater than the amount of label measured in a control application inwhich the cell is not contacted with the test agent. Step (b) may beperformed before step (a).

Other inventive methods comprise steps of: (a) exposing an organism to atest agent; (b) administering to the organism an effective amount of anucleoside analogue that comprises a first reactive unsaturated groupsuch that the nucleoside analogue is incorporated into DNA of cells ofthe organism; (c) contacting at least one cell of the organism with areagent comprising a second reactive unsaturated group attached to alabel, such that a [3+2] cycloaddition occurs between the first andsecond reactive unsaturated groups; (d) determining an amount of labelincorporated into the DNA, wherein the amount of label indicates theextent of cellular proliferation; and (e) identifying the test agent asan agent that perturbs cellular proliferation in the organism if theamount of label measured in step (d) is less than or greater than theamount of label measured in a control application in which the organismis not exposed to the test agent. Step (b) may be performed before step(a). The step of contacting may be performed in vivo or ex vivo.

The inventive methods for identifying an agent that perturb cellularproliferation may further comprise: a step of identifying the test agentas an agent that induces cellular proliferation if the amount of labelmeasured in step (d) is greater than the amount of label measured in thecontrol application; and/or a step of identifying the test agent as anagent that inhibits cellular proliferation if the amount of labelmeasured in step (d) is less than the amount of label measured in thecontrol application.

These and other objects, advantages and features of the presentinvention will be apparent to those of ordinary skill in the art inreading the following detailed description.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 presents two schemes showing (A) DNA labeling using BrdU as knownin the art, and (B) DNA labeling using a method of the present inventionthat includes incorporation of the nucleoside analogue EdU (i.e.,ethynyl-dU) into DNA by DNA replication followed by [3+2] cycloadditionbetween the ethynyl group and the azide reagent in the presence ofCu(I).

FIG. 2 presents a scheme showing an example of 2-color DNA labelingaccording to the present invention.

FIG. 3 presents a scheme showing an example of DAB photoconversionaccording to the present invention.

FIG. 4 presents a scheme illustrating a method of the present inventionfor quenching non-specific (i.e., background) signal from unreactedstaining reagent (i.e., the fluorophore-azide).

FIG. 5 presents two images obtained by fluorescence microscopy of (A)EdU-unlabeled HeLa cells stained with XRhodamine-azide and (B)EdU-labeled cells stained with XRhodamine-azide as described in Example2.

FIG. 6 presents of set of time-lapse fluorescence microscopy imagesshowing the staining reaction on live cells as a function of time.Staining of EdU-labeled cells was performed using a cell-permeableTMR-azide, as described in Example 3. Indications of time on the imagesare given in minutes:seconds.

FIG. 7 is a graph showing the DNA staining intensity plotted over timeas measured on the images presented on FIG. 6.

FIG. 8 shows a fluorescence microscopy image (high magnification) of asection through a mouse intestine labeled as described in Example 4. Thecells with red nuclei are the cells which incorporated EdU and theirdescendants. DNA appears in blue (Hoechst).

FIG. 9 shows a fluorescence microscopy image (low magnification) of asection through a mouse intestine labeled as described in Example 4. DNAappears in green. Red indicates the cells which incorporated EdU andtheir descendants. The object presented on this figure is an entireoblique section through the intestine—about half a centimeter in length.

FIG. 10 shows a fluorescence microscopy image of a section through amouse brain labeled as described in Example 4. As sole EdU-labeled cellcan be easily identified on this brain section (cells of the brainalmost never divide, unlike cells of the intestine, which are highlyproliferative).

FIG. 11 presents two sets of high resolution images of two cells labeledwith EdU. Images (A) and (B) on the left show EdU-labeled cells stainedwith OliGreen, a dye that stains total cellular DNA. Images (C) and (D)on the right show EdU-labeled cells stained with Xrhodamine-azide. Thetop images show the cell in interphase, while the images at the bottomshow the cells in anaphase.

FIG. 12 is a scheme showing the procedure followed to label only one DNAmolecule of the two that form a chromosome.

FIG. 13 is a set of images of the DNA of a EdU-labeled cell stained withAlexa 568-azide as described in Example 5. The first row of images showthe DNA, azide stain and an overlay of the two after the first mitosis.The second row of images show the DNA, azide stain and an overlay of thetwo after the second mitosis.

FIG. 14 is a set of three images demonstrating the use of EU(ethynyl-uridine) as a label for cellular RNA. HeLa cells were labeledwith 10 μM EU overnight, fixed and stained with Xrhodamine-azide. Theimage on the left shows cells that were stained with Xrhodamine-azidewithout having been labeled with EU (negative control). The center andright images are of EU-labeled cells stained with Xrhodamine-azide.

FIG. 15 is a scheme showing ribosome display using a [3+2] cycloadditionaccording to the present invention.

FIG. 16 is a set of two images of a field of HeLa cells labeled with AdUand stained with Alexa568-alkyne (see Example 7 for details). AdU isclearly detected in the nuclei of the labeled interphase cells (A) aswell as in the condensed chromosomes of mitotic cells (B).

FIG. 17 is a set of four images demonstrating a strategy describedherein to effectively remove fluorescent signal from cell nuclei. HeLacells were either left unstained (first row, left hand side); labeledwith EdU and stained with Alexa568-azide (first row, right hand side);labeled with EdU and stained with Alexa568-azide and treated with 20 mMof DTT (second row, left hand side) or 100 mM of DTT (second row, righthand side) (see Example 8 for details).

DEFINITIONS

For purpose of convenience, definitions of a variety of terms usedthroughout the specification are presented below.

The term “nucleic acid polymer” refers to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form,including DNA and RNA, and unless otherwise stated encompasses nucleicacid-like structures with synthetic backbones, as well as amplificationproducts. In the context of the present invention, a nucleic acidpolymer may be an isolated molecule, or, alternatively, a nucleic acidpolymer may be located inside a cell or in an organism.

The term “isolated”, when used herein in reference to a nucleic acidpolymer, means a nucleic acid polymer, which by virtue of its origin ormanipulation is separated from at least some of the components withwhich it is naturally associated or with which it is associated wheninitially obtained. By “isolated”, it is alternatively or additionallymeant that the nucleic acid polymer of interest is produced orsynthesized by the hand of man.

As used herein, the term “nucleotide analogue” refers to a molecule thatis structurally similar to a natural nucleotide and that can function ina similar manner as the naturally occurring nucleotide (e.g., exhibitssimilar ability to base pair with one of the naturally occurring bases).The term “nucleoside analogue”, as used herein, refers to a moleculethat is structurally similar to a natural nucleoside and that canfunction in a similar manner as the naturally occurring nucleoside(e.g., exhibits similar ability to be incorporated into DNA by DNAreplication). The term “nucleotide” refers to a monomeric unit of DNA orRNA containing a sugar moiety (pentose), a phosphate and a nitrogenousheterocyclic base. The base is linked to the sugar moiety via theglycosidic carbon (i.e., the 1′-carbon of the pentose) and thatcombination of base and sugar is called a “nucleoside”. The basecharacterizes the nucleotide (or nucleoside) with the four bases of DNAbeing adenine (or A), guanine (G), cytosine (C) and thymine (T), and thefour bases of RNA being adenine, guanine, cytosine, and uracil (U). Incertain embodiments of the present invention a nucleotide analogue (ornucleoside analogue) comprises a reactive unsaturated group.

As used herein, the term “reactive unsaturated group” refers to afunctional group containing atoms sharing more than one valence bond andthat can undergo addition reactions, in particular cycloadditions. Areactive unsaturated group typically possesses at least one double ortriple bond.

The term “1,3-dipole” has herein its art understood meaning and refersto a molecule or functional group that is isoelectronic with the allylanion and has four electrons in a π system encompassing the 1,3-dipole.1,3-Dipoles generally have one or more resonance structures showing thecharacteristic 1,3-dipole. Examples of 1,3-dipoles include nitrileoxides, azides, diazomethanes, nitrones, and nitrile imines.

As used herein, the term “dipolarophile” has its art understood meaningand refers to a molecule or functional group that contains a π bond andthat exhibits reactivity toward 1,3-dipoles. The reactivity ofdipolarophiles depends both on the substituents present on the π bondand on the nature of the 1,3-dipole involved in the reaction.Dipolarophiles are typically alkenes or alkynes.

As used herein, the term “cycloaddition” refers to a chemical reactionin which two or more π-electron systems (e.g., unsaturated molecules orunsaturated parts of the same molecule) combine to form a cyclic productin which there is a net reduction of the bond multiplicity. In acycloaddition, the π electrons are used to form new σ bonds. The productof a cycloaddition is called an “adduct” or “cycloadduct”. Differenttypes of cycloadditions are known in the art including, but not limitedto, [3+2] cycloadditions and Diels-Alder reactions. [3+2 ]cycloadditions, which are also called 1,3-dipolar cycloadditions, occurbetween a 1,3-dipole and a dipolarophile and are typically used for theconstruction of five-membered heterocyclic rings. The term “[3+2]cycloaddition” also encompasses “copperless” [3+2] cycloadditionsbetween azides and cyclooctynes and difluorocyclooctynes described byBertozzi et al., J. Am. Chem. Soc., 2004, 126: 15046-15047).

The terms “labeled”, “labeled with a detectable agent”, and “labeledwith a detectable moiety” are used herein interchangeably. When used inreference to a nucleic acid polymer, these terms specify that thenucleic acid polymer can be detected or visualized. Preferably, a labelis selected such that it generates a signal which can be measured andwhose intensity is related to the amount of labeled nucleic acidpolymers (e.g., in a sample). In array-based detection methods of theinvention, the label may desirably be selected such that it generates alocalized signal, thereby allowing spatial resolution of the signal fromeach spot on the array. A label may be directly detectable (i.e., itdoes not require any further reaction or manipulation to be detectable,e.g., a fluorophore is directly detectable) or it may be indirectlydetectable (i.e., it is made detectable through reaction or binding withanother entity that is detectable, e.g., a hapten is detectable byimmunostaining after reaction with an appropriate antibody comprising areporter such as a fluorophore). Labels suitable for use in the presentinvention may be detectable by any of a variety of means including, butnot limited to, spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Suitable labelsinclude, but are not limited to, various ligands, radionuclides,fluorescent dyes, chemiluminescent agents, microparticles, enzymes,calorimetric labels, magnetic labels, and haptens.

The terms “fluorophore”, “fluorescent moiety” and “fluorescent dye” areused herein interchangeably. They refer to a molecule which, in solutionand upon excitation with light of appropriate wavelength, emits lightback, generally at a longer wavelength. Numerous fluorescent dyes of awide variety of structures and characteristics are suitable for use inthe practice of the present invention. In choosing a fluorophore, it isoften desirable that the molecule absorbs light and emits fluorescencewith high efficiency (i.e., the fluorescent molecule has a high molarextinction coefficient at the excitation wavelength and a highfluorescence quantum yield, respectively) and is photostable (i.e., thefluorescent molecule does not undergo significant degradation upon lightexcitation within the time necessary to perform the detection).

As used herein, the term “dual labeling” refers to a labeling process inwhich a nucleic acid polymer is labeled with two detectable agents thatproduce distinguishable signals. The nucleic acid polymer resulting fromsuch a labeling process is said to be dually labeled. As used herein,the term “differential labeling” refers to a labeling process in whichtwo nucleic acid polymers are labeled with two detectable agents thatproduce distinguishable signals (i.e., a first nucleic acid polymer islabeled with a first detectable agent, a second nucleic acid polymer islabeled with a second detectable agent, and the first and seconddetectable agents produce distinguishable signals). The detectableagents may be of the same type (e.g., two fluorescent dyes that producedual-color fluorescence upon excitation) or of different types (e.g., afluorescent dye and a hapten).

The terms “cell proliferation” and “cellular proliferation” are usedherein interchangeably and refer to an expansion of a population ofcells by the division of single cells into daughter cells, or to thedivision of a single cell to daughter cells.

As used herein, the term “effective amount” refers to the amount of asubstance, compound, molecule, agent or composition that elicits therelevant response in a cell, a tissue, or an organism. For example, inthe case of a nucleoside administered to an organism, an effectiveamount of nucleoside is an amount of nucleoside that is incorporatedinto the DNA of cells of the organism.

As used herein, the term “organism” refers to a living system that hasor can develop the ability to act or function independently. An organismmay be unicellular or multicellular. Organisms include humans, animals,plants, bacteria, protozoa, and fungi.

The term “perturbation of cellular proliferation”, as used herein,refers to the ability of an agent to induce (i.e., increase, enhance orotherwise exacerbate) or inhibit (i.e., decrease, slow down or otherwisesuppress) cell proliferation as compared to cellular proliferationobserved in the absence of the agent.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

As mentioned above, the present invention provides methods andcompositions for labeling nucleic acid polymers and for measuringcellular proliferation both in vitro and in vivo.

I. Labeling of Nucleic Acid Polymers

The labeling methods of the present invention generally include a [3+2]cycloaddition between a first reactive unsaturated group on a nucleotideincorporated into a nucleic acid polymer and a second reactiveunsaturated group attached to a label. An example of such a labelingmethod is schematically presented on FIG. 1 and compared to theconventional BrdU labeling method.

1. Nucleoside and Nucleotide Analogues

Nucleoside analogues (or nucleotide analogues) suitable for use in thepractice of the methods of the present invention include any nucleosideanalogue (or nucleotide analogue), as defined herein, that contains areactive unsaturated group that can undergo a [3+2] cycloaddition. Insome embodiments, the reactive unsaturated group is carried by the baseof the nucleoside (or nucleotide). The base carrying the reactiveunsaturated group can be a purine (e.g., adenine or guanine) or apyrimidine (e.g., cytosine, uracil or thymine). In certain embodiments,the base is uracil; in some such embodiments, uracil carries thereactive unsaturated group on the 5-position. The unsaturated group canbe directly or indirectly covalently attached to the base. Preferably,the unsaturated group is directly covalently attached to the base.

The reactive unsaturated group can be a 1,3-dipole such as a nitrileoxide, an azide, a diazomethane, a nitrone or a nitrile imine. Incertain embodiments, the 1,3-dipole is an azide. Alternatively, thereactive unsaturated group can be a dipolarophile such as an alkene(e.g., vinyl, propylenyl, and the like) or an alkyne (e.g., ethynyl,propynyl, and the like). In certain embodiments, the dipolarophile is analkyne, such as, for example, an ethynyl group.

Methods for the preparation of nucleoside analogues and nucleosidetriphosphate analogues are known in the art. For example, procedures forthe preparation of 5-substituted bases in nucleosides and nucleosidetriphosphates have been developed and reported (see, for example, A. S.Jones et al., Nucleic Acids Res., 1974, 1: 105-107; R. C. Bleackley etal., Nucleic Acids Res., 1975, 2: 683-690; D. E. Bergstrom and J. L.Ruth, J. Am. Chem. Soc., 1976, 98: 1587-1589; Y. F. Shealy et al., J.Med. Chem., 1983, 26: 156-161; K. He et al., Nucleic Acids Res., 1999,8:1788-1798; H. A. Held and S. A. Benner, Nucleic Acids Res., 2002, 30:3857-3869).

Exemplary nucleoside analogues that may be used in the practice of thepresent invention include 5-ethynyl-2′deoxyuracil (also termed hereinethynyluracil or EdU) and 5-azido-2′-deoxyuracil (also termed hereinazidouracil or AdU) as well as their triphosphate and phosphoramiditeforms. The present Applicants synthesized EdU essentially as describedby C.-S. Yu and F. Oberdorfer, Synlett, 2000, 1: 86-88; and prepared AdUusing a method similar to that described in P. Sunthankar et al., Anal.Biochem., 1998, 258: 195-201 to synthesize azido-dUMP. EdU is alsocommercially available from Berry and Associates, Inc. (Dexter, Mich.).

2. Nucleic Acid Polymers

Nucleic acid polymers produced according to methods of the presentinvention or utilized in methods of the present invention are single- ordouble-stranded deoxyribonucleotide or ribonucleotide polymers. As willbe appreciated by one of ordinary skill in the art, the nucleic acidpolymers can be polynucleotides of any of a wide range of sizesincluding short oligonucleotides comprising at least about 8 nucleotidesas well as full genomic DNA molecules.

Nucleic acid polymers containing at least one nucleotide analogue may beprepared by any of a variety of methods well known in the art includingsynthetic and enzymatic methods (J. Sambrook et al., “Molecular Cloning:A Laboratory Manual”, 1989, 2^(nd) Ed., Cold Spring Harbour LaboratoryPress: New York, N.Y.; “PCR Protocols: A Guide to Methods andApplications”, 1990, M. A. Innis (Ed.), Academic Press: New York, N.Y.;P. Tijssen “Hybridization with Nucleic Acid Probes—Laboratory Techniquesin Biochemistry and Molecular Biology (Parts I and II)”, 1993, ElsevierScience; “PCR Strategies”, 1995, M. A. Innis (Ed.), Academic Press: NewYork, N.Y.; and “Short Protocols in Molecular Biology”, 2002, F. M.Ausubel (Ed.), 5^(th) Ed., John Wiley & Sons: Secaucus, N.J.).

For example, the inventive nucleic acid polymers may be prepared usingautomated, solid-phase procedure based on the phosphoramidite approach.In such a method, each nucleotide (including nucleotide analogues) isindividually added to the 5′-end of the growing polynucleotide chain,which is attached at the 3′-end to a solid support. The addednucleotides are in the form of trivalent 3′-phosphoramidites that areprotected from polymerization by a dimethoxytriyl (or DMT) group at the5′-position. After base-induced phosphoramidite coupling, mild oxidationto give a pentavalent phosphotriester intermediate, DMT removal providesa new site for polynucleotide elongation. The nucleic acid polymers arethen cleaved off the solid support, and the phosphodiester and exocyclicamino groups are deprotected with ammonium hydroxide. These synthesesmay be performed on oligo synthesizers such as those commerciallyavailable from Perkin Elmer/Applied Biosystems, Inc (Foster City,Calif.), DuPont (Wilmington, Del.) or Milligen (Bedford, Mass.).

The inventive nucleic acid polymers can alternatively be prepared, forexample using in vitro extension and/or amplification methods. Standardnucleic acid amplification methods include: polymerase chain reaction orPCR (“PCR Protocols: A Guide to Methods and Applications”, M. A. Innis(Ed.), Academic Press: New York, 1990; and “PCR Strategies”, M. A. Innis(Ed.), Academic Press: New York, 1995); ligase chain reaction or LCR (U.Landegren et al., Science, 1988, 241: 1077-1080; and D. L. Barringer etal., Gene, 1990, 89: 117-122); transcription amplification (D. Y. Kwohet al., Proc. Natl. Acad. Sci. USA, 1989, 86: 1173-1177); self-sustainedsequence replication (J. C. Guatelli et al., Proc. Natl. Acad. Sci. USA,1990, 87: 1874-1848); Q-beta replicase amplification (J. H. Smith etal., J. Clin. Microbiol. 1997, 35: 1477-1491); automated Q-betareplicase amplification assay (J. L. Burg et al., Mol. Cell. Probes,1996, 10: 257-271) and other RNA polymerase mediated techniques such as,for example, nucleic acid sequence based amplification or NASBA (A. E.Greijer et al., J. Virol. Methods, 2001, 96: 133-147).

As will be appreciated by one of ordinary skill in the art, nucleic acidpolymers of the present invention may be prepared either by apre-synthetic modification method (i.e., incorporation of nucleotidesanalogues into the nucleic acid molecule) or a post-syntheticmodification method (i.e., modification of naturally occurringnucleotides to nucleotide analogues in the nucleic acid molecule).

Alternatively, nucleotide analogues can be incorporated into the DNA ofcells or living systems by DNA replication, or into RNA by reaction, asdescribed below.

Isolation or purification of the nucleic acid polymers of the presentinvention, where necessary, may be carried out by any of a variety ofmethods well-known in the art. Purification of nucleic acid polymers istypically performed either by native acrylamide gel electrophoresis, byanion-exchange HPLC as described, for example by J. D. Pearson and F. E.Regnier (J. Chrom., 1983, 255: 137-149) or by reverse phase HPLC (G. D.McFarland and P. N. Borer, Nucleic Acids Res., 1979, 7: 1067-1080)

If desired, the sequence of synthetic nucleic acid polymers can beverified using any suitable sequencing method including, but not limitedto, chemical degradation (A. M. Maxam and W. Gilbert, Methods ofEnzymology, 1980, 65: 499-560), matrix-assisted laser desorptionionization time-of-flight (MALDI-TOF) mass spectrometry (U. Pieles etal., Nucleic Acids Res., 1993, 21: 3191-3196), mass spectrometryfollowing alkaline phosphatase and exonuclease digestions (H. Wu and H.Aboleneen, Anal. Biochem., 2001, 290: 347-352), and the like.

3. [3+2] Cycloaddition

The methods provided herein generally include a [3+2] cycloaddition. Inthese methods, the [3+2] cycloaddition occurs between a first reactiveunsaturated group on a nucleotide analogue incorporated into a nucleicacid polymer and a second reactive unsaturated group on a reagentcomprising a label (also called herein a staining reagent).

In some embodiments of the present invention, the staining reagent isselected such that the second reactive unsaturated group can react via a[3+2] cycloaddition with the first reactive unsaturated group on thenucleotide analogue. More specifically, if the first unsaturated groupis a 1,3-dipole, the second unsaturated group will be a dipolarophilethat can react with the 1,3-dipole. Alternatively, if the firstunsaturated group is a dipolarophile, the second unsaturated group willbe a 1,3-dipole that can react with the dipolarophile.

Optimization of [3+2] cycloaddition reaction conditions is within theskill of the art. In certain preferred embodiments, the [3+2]cycloaddition is performed under aqueous conditions.

In embodiments where the 1,3-dipole is an azide and the dipolarophile isan alkyne (e.g., ethynyl group), the [3+2] cycloaddition may beperformed as described by Sharpless and coworkers (V. V. Rostovtsev etal., Angew Chem., Int. Ed. Engl., 2002, 41: 1596-1599; W. G. Lewis etal., Angew Chem. Int. Ed. Engl., 2002, 41: 1053-1057; Q. Wang et al., J.Am. Chem. Soc., 2003, 125: 3192-3193) at physiological temperatures,under aqueous conditions and in the presence of copper(I) (or Cu(I)),which catalyzes the cycloaddition. This catalyzed version of the [3+2]cycloaddition is termed “click” chemistry.

In other embodiments, for example where the presence of exogenous Cu(I)is not desired (e.g., when Cu(I) is toxic to a living system), the [3+2]cycloaddition between the azide and the alkyne may be performed asdescribed by Sharpless and coworkers except for the presence of Cu(I).In these situations, the staining reagent used in the cycloadditioncomprises a Cu chelating moiety in addition to a reactive unsaturatedgroup and a label. As used herein, the term “Cu chelating moiety” refersto any entity characterized by the presence of two or more polar groupsthat can participate in the formation of a complex (containing more thanone coordinate bond) with copper(I) ions. A Cu chelating moiety canmobilize copper(I) ions naturally present in a living system (e.g., acell) in the vicinity of the [3+2] cycloaddition. Specific Cu(I)chelators are known in the art and include, but are not limited to,neocuproine (H. H. Al-Sa'doni et al., Br. J. Pharmacol., 1997, 121:1047-1050; J. G. De Man et al., Eur. J. Pharmacol., 1999, 381: 151-159;C. Gocmen et al., Eur. J. Pharmacol., 2000, 406: 293-300) andbathocuproine disulphonate (M. Bagnati et al., Biochem. Biophys. Res.Commun., 1998, 253: 235-240).

4. Labels and Detection of Labeled Nucleic Acid Polymers

The methods of the present invention include a [3+2] cycloadditionbetween a first reactive unsaturated group on a nucleotide analogueincorporated into a nucleic acid polymer and a second reactiveunsaturated group attached to a label. The [3+2] cycloaddition reactionresults in labeling of the nucleic aid polymer.

A. Labels

As already mentioned above, the role of a label is to allowvisualization or detection of a nucleic acid polymer, e.g., DNA in acell, following labeling. Preferably, a label (or detectable agent ormoiety) is selected such that it generates a signal which can bemeasured and whose intensity is related (e.g., proportional) to theamount of labeled nucleic acid polymer, e.g., in a sample beinganalyzed. In array-based detection methods of the invention (see below),the detectable agent is also preferably selected such that it generatesa localized signal, thereby allowing spatial resolution of the signalfor each spot on the array.

The association between the label and the staining reagent comprisingthe second reactive unsaturated group is preferably covalent. A labelcan be directly attached to the unsaturated group on the stainingreagent or indirectly through a linker.

Methods for attaching detectable moieties to chemical molecules arewell-known in the art. In certain embodiments, the label and unsaturatedgroup are directly, covalently linked to each other. The direct covalentbinding can be through an amide, ester, carbon-carbon, disulfide,carbamate, ether, thioether, urea, amine, or carbonate linkage. Thecovalent binding can be achieved by taking advantage of functionalgroups present on the unsaturated group and detectable moiety. Suitablefunctional groups that can be used to attach the two chemical entitiestogether include, but are not limited to, amines, anhydrides, hydroxygroups, carboxy groups, and thiols. A direct linkage may also be formedusing an activating agent, such as a carbodiimide. A wide range ofactivating agents are known in the art and are suitable for linking alabel and an unsaturated group.

In other embodiments, the unsaturated group of the staining reagent andthe label are indirectly covalently linked to each other via a linkergroup. This can be accomplished by using any number of stablebifunctional agents well known in the art, including homofunctional andheterofunctional linkers (see, for example, Pierce Catalog andHandbook). The use of a bifunctional linker differs from the use of anactivating agent in that the former results in a linking moiety beingpresent in the reaction product, whereas the latter results in a directcoupling between the two moieties involved in the reaction. The role ofthe bifunctional linker may be to allow the reaction between twootherwise inert moieties. Alternatively or additionally, thebifunctional linker, which becomes part of the reaction product, may beselected such that it confers some degree of conformational flexibilityto the reaction product, or other useful or desired properties. Incertain embodiments, the linker is cleavable (e.g., chemically cleavableor photochemically cleavable). The presence of a cleavable linkerbetween the label and the nucleotide analogue allows for temporarylabeling of the nucleic acid polymer. With such a system, wheneverdesired (e.g., following detection of the nucleic acid polymer), thelabel can be cleaved off the nucleotide analogue to which it isattached. Cleavable linkers are known in the art. For example, thelinker may be a cystamine linker, the disulfide bond of which can bereduced using dithiothreitol (DTT) (see Example 8).

Any of a wide variety of labeling/detectable agents can be used in thepractice of the present invention. Suitable detectable agents include,but are not limited to, various ligands, radionuclides (such as, forexample, ³²P, ³⁵S, ³H, ¹⁴C, ¹²⁵I, ¹³¹I, and the like); fluorescent dyes(for specific exemplary fluorescent dyes, see below); chemiluminescentagents (such as, for example, acridinium esters, stabilized dioxetanes,and the like); spectrally resolvable inorganic fluorescent semiconductornanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold,silver, copper and platinum) or nanoclusters; enzymes (such as, forexample, those used in an ELISA, i.e., horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase); colorimetriclabels (such as, for example, dyes, colloidal gold, and the like);magnetic labels (such as, for example, Dynabeads™); and biotin,dioxigenin, haptens, and proteins for which antisera or monoclonalantibodies are available.

In certain embodiments, the label comprises a fluorescent moiety.Numerous known fluorescent labeling moieties of a wide variety ofchemical structures and physical characteristics are suitable for use inthe practice of the present invention. Suitable fluorescent dyesinclude, but are not limited to, fluorescein and fluorescein dyes (e.g.,fluorescein isothiocyanine or FITC, naphthofluorescein,4′,5′-dichloro-2′,7′-dimethoxy-fluorescein, 6-carboxyfluorescein orFAM), carbocyanine, merocyanine, styryl dyes, oxonol dyes,phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g.,carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G,carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G,rhodamine Green, rhodamine Red, tetramethylrhodamine or TMR), coumarinand coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin,hydroxycoumarin and aminomethylcoumarin or AMCA), Oregon Green Dyes(e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514), Texas Red,Texas Red-X, Spectrum Red™, Spectrum Green™, cyanine dyes (e.g. Cy-3™,Cy-5™, Cy-3.5™, Cy-5.5™), Alexa Fluor dyes (e.g., Alexa Fluor 350, AlexaFluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, AlexaFluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), BODIPYdyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591,BODIPY 630/650, BODIPY 650/665), IRDyes (e.g., IRD40, IRD 700, IRD 800),and the like. For more examples of suitable fluorescent dyes and methodsfor coupling fluorescent dyes to other chemical entities see, forexample, “The Handbook of Fluorescent Probes and Research Products”,9^(th) Ed., Molecular Probes, Inc., Eugene, Oreg.

Favorable properties of fluorescent labeling agents to be used in thepractice of the invention include high molar absorption coefficient,high fluorescence quantum yield, and photostability. In certainembodiments, labeling fluorophores desirably exhibit absorption andemission wavelengths in the visible (i.e., between 400 and 750 nm)rather than in the ultraviolet range of the spectrum (i.e., lower than400 nm). Other desirable properties of the fluorescent moiety mayinclude cell permeability and low toxicity, for example if labeling ofthe nucleic acid polymer is to be performed in a cell or an organism(e.g., a living animal).

As reported in the Examples, various fluorescent staining reagents havebeen used by the present Applicants, including XRhodamine-azide andAlexa568-azide, which are non-cell permeable, and tetramethylrhodamine(TMR)-azide, which is cell permeable.

The present invention also provides for two-color labeling of nucleicacid polymers (see FIG. 2). For example, according to the presentinvention, two or more different labels may be incorporated into asingle nucleic acid polymer. In some embodiments, such incorporation isachieved via two [3+2] cycloaddition reactions: a first cycloadditiontakes place between a first reactive unsaturated group on a nucleotideanalogue incorporated into a first nucleic acid polymer and a secondreactive unsaturated group on a first reagent attached to a first label;a second cycloaddition takes place between a third reactive unsaturatedgroup on a nucleotide analogue incorporated into a second nucleic acidpolymer and a fourth reactive unsaturated group on a second reagentattached to a second label. The first and second nucleic acid polymersmay be the same molecule (e.g., dual labeling of DNA in a cell) ordifferent/individual molecules (e.g., differential labeling of DNA fromtwo different cells, cell populations or cell samples). The first andsecond labels are preferably selected such that they producedistinguishable detectable signals.

In certain two-label embodiments, the first and second detectable agentsor labels are fluorescent dyes. To allow for two-color detection, thefirst and second fluorescent labels may desirably constitute a matchedpair that is compatible with the detection system to be used. Matchedpairs of fluorescent labeling dyes typically produce signals that arespectrally distinguishable. For example, in some embodiments, thefluorescent dyes in a matched pair do not significantly absorb light inthe same spectral range (i.e., they exhibit different absorption maximawavelengths) and can be excited (for example, sequentially) using twodifferent wavelengths. Alternatively, the fluorescent dyes in a matchedpair may emit light in different spectral ranges (i.e., they produce adual-color fluorescence upon excitation).

Pairs of fluorescent labels are known in the art (see, for example, R.P. Haugland, “Molecular Probes: Handbook of Fluorescent Probes andResearch Chemicals 1992-1994”, 5^(th) Ed., 1994, Molecular Probes,Inc.). Exemplary pairs of fluorescent dyes include, but are not limitedto, rhodamine and fluorescein (see, for example, J. DeRisi et al.,Nature Gen., 1996, 14: 458-460); Spectrum Red™ and Spectrum Green(commercially available from Vysis, Inc., Downers Grove, Ill.); andCy-3™ and Cy-5 (commercially available from Amersham Life Sciences,Arlington Heights, Ill.).

The selection of a particular label (or set of labels) will depend onthe purpose of the labeling to be performed and will be governed byseveral factors, such as the ease and cost of the labeling method, thequality of sample labeling desired, the effects of the detectable moietyon the cell or organism, the nature of the detection system, the natureand intensity of the signal generated by the detectable moiety, and thelike.

B. Detection of Labeled Nucleic Acid Polymers

As will be recognized by one of ordinary skill in the art, detection ofnucleic acid polymers labeled according to methods disclosed herein maybe performed by any of a wide variety of methods, and using any of awide variety of techniques. Selection of a suitable detection methodand/or detection technique based on the nature of the label (e.g.,radionuclide, fluorophore, chemiluminescent agent, quantum dot, enzyme,magnetic label, hapten, etc) is within the skill in the art.

For example, fluorescently labeled nucleic acid polymers may be detectedusing fluorescence detection techniques, including, but not limited to,flow cytometry and fluorescence microscopy. Selection of a specificfluorescence detection technique will be governed by many factorsincluding the purpose of the labeling experiment (e.g., study ofchromosomes ultrastructure, cell proliferation determination, ortoxicity assay) as well as the location of the labeled nucleic acidpolymer to be detected (i.e., such as inside a living cell or inside atissue).

Flow cytometry is a sensitive and quantitative technique that analyzesparticles (such as cells) in a fluid medium based on the particles'optical characteristic (H. M. Shapiro, “Practical Flow Cytometry”,3^(rd) Ed., 1995, Alan R. Liss, Inc.; and “Flow Cytometry and Sorting,Second Edition”, Melamed et al. (Eds), 1990, Wiley-Liss: New York). Aflow cytometer hydrodynamically focuses a fluid suspension of particlescontaining one or more fluorophores, into a thin stream so that theparticles flow down the stream in a substantially single file and passthrough an examination or analysis zone. A focused light beam, such as alaser beam, illuminates the particles as they flow through theexamination zone, and optical detectors measure certain characteristicsof the light as it interacts with the particles (e.g., light scatter andparticle fluorescence at one or more wavelengths).

Alternatively or additionally, fluorescently labeled nucleic acidpolymers in cells, tissues or organisms may be visualized and detectedby fluorescence microscopy using different imaging techniques. Inaddition to conventional fluorescence microscopy, fluorescently labelednucleic acid polymers can be analyzed by, for example, time-lapsefluorescence microscopy, confocal fluorescence microscopy, or two-photonfluorescence microscopy. Time-lapse microscopy techniques (D. J.Stephens and V. J. Allan, Science, 2003, 300: 82-86) can provide acomplete picture of complex cellular processes that occur in threedimensions over time. Information acquired by these methods allowdynamic phenomena such as cell growth, cell motion and cell nucleidivision to be monitored and analyzed quantitatively. Confocalmicroscopy (L. Harvath, Methods Mol. Biol., 1999, 115: 149-158; Z.Foldes-Papp et al., Int. Immunopharmacol., 2003, 3: 1715-1729) offersseveral advantages over conventional optical microscopy, includingcontrollable depth of field, the elimination of image degradingout-of-focus information, and the ability to collect serial opticalsections from thick specimens (e.g., tissues or animals). Two-photonfluorescence microscopy (P. T. So et al., Annu. Rev. Biomed. Eng., 2000,2; 399-429), which involves simultaneous absorption of two photons bythe fluorophore at the focal point of the microscope, allowsthree-dimensional imaging in highly localized volumes (e.g., in thenucleus of cells) with minimal photobleaching and photodamage.

Signals from fluorescently labeled nucleic acid polymers attached tomicroarrays or located inside cells in multi-well plates can be detectedand quantified by any of a variety of automated and/or high-throughputinstrumentation systems including fluorescence multi-well plate readers,fluorescence activated cell sorters (FACS) and automated cell-basedimaging systems that provide spatial resolution of the signal. Methodsfor the simultaneous detection of multiple fluorescent labels and thecreation of composite fluorescence images are well-known in the art andinclude the use of “array reading” or “scanning” systems, such ascharge-coupled devices (i.e., CCDs) (see, for example, Y. Hiraoka etal., Science, 1987, 238: 36-41; R. S. Aikens et al., Meth. Cell Biol.1989, 29: 291-313; A. Divane et al., Prenat. Diagn. 1994, 14: 1061-1069;S. M. Jalal et al., Mayo Clin. Proc. 1998, 73: 132-137; V. G. Cheung etal., Nature Genet. 1999, 21: 15-19; see also, for example, U.S. Pat.Nos. 5,539,517; 5,790,727; 5,846,708; 5,880,473; 5,922,617; 5,943,129;6,049,380; 6,054,279; 6,055,325; 6,066,459; 6,140,044; 6,143,495;6,191,425; 6,252,664; 6,261,776; and 6,294,331). A variety ofinstrumentation systems have been developed to automate such analysesincluding the automated fluorescence imaging and automated microscopysystems developed by Cellomics, Inc. (Pittsburgh, Pa.), AmershamBiosciences (Piscataway, N.J.), TTP LabTech Ltd (Royston, UK),Quantitative 3 Dimensional Microscopy (Q3DM) (San Diego, Calif.), EvotecAG (Hamburg, Germany), Molecular Devices Corp. (Sunnyvale, Calif.), andCarl Zeiss AG (Oberkochen, Germany).

The fluorescent signal from labeled nucleic acid polymers within a cellor a tissue can also be visualized after DAB (diaminobenzidine)photoconversion (as shown on FIG. 3). Photoconversion is the process bywhich a fluorescent probe (e.g., within a cell) is converted into anelectron-dense probe, labeling the area or structure of interest forstudy at light and electron microscope levels. The principle ofphotoconverting fluorescent dyes to an insoluble light andelectron-dense diaminobenzidine (DAB) reaction product was firstdemonstrated by A. R. Maranto (Science, 1982, 217: 953-955). DABphotoconversion is generally accomplished by incubating thefluorescently labeled cells with or bathing the fluorescently labeledtissue in a DAB solution while exposing the cells or tissue to afrequency of light that maximally excites the fluorophore.

A diaminobenzidine stain may have several advantages over a fluorescentstain due to its greater stability and greater density Adiaminobenzidine stain can also be intensified by employing osmiumtetroxide and potassium ferrocyamide following the DAB treatment (H. C.Mutasa, Biotech. Histochem., 1995, 70: 194-201). Examples offluorophores that have been reported to undergo efficient DAB conversioninclude, but are not limited to, 4,6-diamidino-2-phenylindole (DAPI),Fast Blue, Lucifer Yellow, Diamidino Yellow, Evans Blue, acridineorange, ethidium bromide, 5,7-dihydroxytryptamine,1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indolcarbocyanine perchlorate,3,3′-dioctadecylindocarbocyanine (DiI), rhodamine-123, and propidiumiodide.

Before DAB photoconversion, fluorescently labeled nucleic acid polymersin cells or tissue may be detected and localized by fluorescencemicroscopy. After DAB conversion, the nucleic acid polymers may bevisualized by transmission electron microscopy.

C. Signal-to-Noise Ratio Improvements

In another aspect, the present invention provides a system for improvingthe signal-to-noise ratio in the detection of a nucleic acid polymerlabeled with a fluorescent moiety using a labeling process disclosedherein.

Any molecule of staining reagent that has not been consumed by the [3+2]cycloaddition labeling reaction may contribute to the background (i.e.,non-specific) signal. The present invention provides a strategy forreducing or eliminating this background signal which comprises quenchingthe fluorescent signal of the label on the unreacted staining reagent byreaction with a molecule comprising a quencher moiety. For example, insome embodiments, the reaction between the reagent and the moleculecomprising the quencher moiety is a [3+2] cycloaddition (as shown onFIG. 4).

Thus, certain inventive methods for improving the signal-to-noise ratioin detection of a fluorescently labeled nucleic acid polymer prepared asdescribed herein, comprise contacting unreacted reagent comprising asecond reactive unsaturated group and a fluorescent label (andoptionally a Cu chelating moiety) with a quenching molecule comprising areactive unsaturated group attached to a quenching moiety such that a[3+2] cycloaddition takes place between the reactive unsaturated groupsof the staining reagent and quenching molecule. After reaction, thephysical proximity between the fluorescent label and the quenchingmoiety prevents detection of a fluorescent signal from the fluorescentlabel.

Examples of quenching moieties include, but are not limited to DABCYL(i.e., 4-(4′-dimethylaminophenylazo)-benzoic acid) succinimidyl ester,diarylrhodamine carboxylic acid, succinimidyl ester (or QSY-7), and4′,5′-dinitrofluorescein carboxylic acid, succinimidyl ester (or QSY-33)(all available, for example, from Molecular Probes), quencher1 (Q1;available from Epoch Biosciences, Bothell, Wash.), or “Black holequenchers” BHQ-1, BHQ-2, and BHQ-3 (available from BioSearchTechnologies, Inc., Novato, Calif.).

5. Labeling of Nucleic acid Polymers in Cells

The present invention also provides methods for labeling nucleic acidpolymers in cells. Such methods comprise: contacting a cell with aneffective amount of a nucleoside analogue that comprises a firstreactive unsaturated group such that the nucleoside analogue isincorporated into DNA of the cell; contacting the cell with a reagentcomprising a second reactive unsaturated group attached to a label suchthat a [3+2] cycloaddition occurs between the first and second reactiveunsaturated groups.

Unless otherwise stated, the staining reagent and [3+2] cycloadditionconditions used in these methods are analogous to those described abovefor the methods of labeling nucleic acid polymers. As already mentionedabove, the labeling methods of the present invention exhibit severaladvantages over currently available labeling protocols including thepossibility of staining nucleic acid polymers in living cells. The terms“living cell” and “live cell” are used herein interchangeably and referto a cell which is considered living according to standard criteria forthat particular type of cell, such as maintenance of normal membranepotential, energy metabolism, or proliferative capability. Inparticular, the methods of the present invention do not require fixationand/or denaturation of the cells.

A. Cells

In some embodiments, the invention relates to incorporation of labelsinto nucleic acid polymers in cells in culture. In certain embodiments,the cells are grown in standard tissue culture plastic ware. Such cellsinclude normal and transformed cells derived. In certain embodiments,cells are of mammalian (human or animal, such as rodent or simian)origin. Mammalian cells may be of any fluid, organ or tissue origin(e.g., blood, brain, liver, lung, heart, bone, and the like) and of anycell types (e.g., basal cells, epithelial cells, platelets, lymphocytes,T-cells, B-cells, natural killer cells, macrophages, tumor cells, andthe like).

Cells suitable for use in the methods of the present invention may beprimary cells, secondary cells or immortalized cells (i.e., establishedcell lines). They may have been prepared by techniques well-known in theart (for example, cells may be obtained by drawing blood from a patientor a healthy donor) or purchased from immunological and microbiologicalcommercial resources (for example, from the American Type CultureCollection, Manassas, Va.). Alternatively or additionally, cells may begenetically engineered to contain, for example, a gene of interest suchas a gene expressing a growth factor or a receptor.

Cells to be used in the methods of the present invention may be culturedaccording to standard culture techniques. For example, cells are oftengrown in a suitable vessel in a sterile environment at 37° C. in anincubator containing a humidified 95% air-5% CO₂ atmosphere. Vessels maycontain stirred or stationary cultures. Various cell culture media maybe used including media containing undefined biological fluids such asfetal calf serum. Cell culture techniques are well known in the art, andestablished protocols are available for the culture of diverse celltypes (see, for example, R. I. Freshney, “Culture of Animal Cells: AManual of Basic Technique”, 2^(nd) Edition, 1987, Alan R. Liss, Inc.).

B. Incorporation of Nucleoside Analogue by DNA Replication

Incorporation of nucleoside analogues into DNA by DNA replication is aprocess well-known in the art. In general, nucleoside analogues aretransported across the cell membrane by nucleoside transporters and arephosphorylated in cells by kinases to their triphosphate forms. Thenucleoside analogue triphosphates then compete with thenaturally-occurring deoxyribonucleotides as substrates of cellular DNApolymerases. Such a process is used for the incorporation of³H-thymidine and 5′-bromo-2′-deoxyuridine (BrdU) into DNA for labelingpurposes as well as in cancer therapy (D. Kufe et al., Blood, 1984, 64:54-58; E. Beutler, Lancet, 1992, 340: 952-956; Y. F. Hui and J. Reitz,Am. J. Health-Syst. Pharm., 1997, 54: 162-170; H. Iwasaki et al., Blood,1997, 90: 270-278) and in the treatment of human immunodeficiency virusinfection (J. Balzarini, Pharm. World Sci., 1994, 16: 113-126).

Contacting the cells in vitro with an effective amount of a nucleosideanalogue such that the nucleoside analogue is incorporated into DNA ofthe cell may be carried out using any suitable protocol. In certainpreferred embodiments, the nucleoside analogue is incorporated into DNAusing exponentially growing cells or cells in the S-phase of the cellcycle (i.e., the synthesis phase). If desired, cells may be synchronizedin early S-phase by serum deprivation before the labeling-pulseprocedure.

The step of contacting a cell with an effective amount of a nucleosideanalogue may be performed, for example, by incubating the cell with thenucleoside analogue under suitable incubation conditions (e.g., inculture medium at 37° C. as described in Example 1). In certainsituations, it may be desirable to avoid disturbing the cells in any way(e.g., by centrifugation steps or temperature changes) that may perturbtheir normal cell cycling patterns. The incubation time will bedependent on the cell population's rate of cell cycling entry andprogression. Optimization of incubation time and conditions is withinthe skill in the art.

C. [3+2] Cycloaddition in Cells

Following incorporation of the nucleotide analogue into the DNA of invitro cells, the step of contacting the cells with a staining reagentcomprising the second reactive unsaturated group and a label may beperformed by any suitable method. In some embodiments, the cells areincubated in the presence of the staining reagent in a suitableincubation medium (e.g., culture medium) at 37° C. and for a timesufficient for the reagent to penetrate into the cell and react with anynucleotide analogue incorporated into the DNA of the cells. Optimizationof the concentration of staining reagent, cycloaddition reaction timeand conditions is within the skill in the art.

As already mentioned above, in embodiments where the presence ofexogenous Cu(I) is not desirable, the [3+2] cycloaddition may be carriedout using a staining reagent that comprises the second reactiveunsaturated group, a label, and a Cu(I) chelating moiety.

In embodiments where the staining reagent does not exhibit high cellpermeability, permeabilization may be performed to facilitate access ofthe staining reagent to cellular cytoplasm, or intracellular componentsor structures of the cells. In particular, permeabilization may allow areagent to enter into a cell and reach a concentration within the cellthat is greater than that which would normally penetrate into the cellin the absence of such permeabilization treatment.

Permeabilization of the cells may be performed by any suitable method(see, for example, C. A. Goncalves et al., Neurochem. Res. 2000, 25:885-894). Such methods include, but are not limited to, exposure to adetergent (such as CHAPS, cholic acid, deoxycholic acid, digitonin,n-dodecyl-β-D-maltoside, lauryl sulfate, glycodeoxycholic acid,n-lauroylsarcosine, saponin, and triton X-100) or to an organic alcohol(such as methanol and ethanol). Other permeabilization methods comprisethe use of certain peptides or toxins that render membranes permeable(see, for example, O. Aguilera et al., FEBS Lett. 1999, 462: 273-277; A.Bussing et al., Cytometry, 1999, 37: 133-139). Selection of anappropriate permeabilizing agent and optimization of the incubationconditions and time can easily be performed by one of ordinary skill inthe art.

As described in Example 2, the present Applicants have incubated HeLacells in the presence or absence of EdU, permeabilized the cells, andstained them with Xrhodamine-azide. Fluorescence images of the two cellpopulations are presented on FIG. 5. This figure illustrates theefficiency of the inventive labeling methods in vitro. Furthermore,detection of labeled DNA in living cells using such methods was found tobe complete within minutes (see FIG. 6 and FIG. 7). Applicants have alsoincubated HeLa cells in the presence of AdU, and stained them withAlexa568-alkyne (see Example 7). Fluorescence images of cell populationsthus treated are presented on FIG. 16.

6. Labeling of Nucleic acid Polymers in Tissues or Organisms

The present invention also provides methods for labeling nucleic acidpolymers in organisms (i.e., living biological systems). Such methodscomprise steps of: administering to an organism an effective amount of anucleoside analogue that comprises a first reactive unsaturated groupsuch that the nucleoside analogue is incorporated into the DNA in cellsof the organism; contacting at least one cell of the organism with areagent comprising a second reactive unsaturated group attached to alabel such that a [3+2] cycloaddition occurs between the first andsecond reactive unsaturated groups.

Unless otherwise stated, the staining reagents and [3+2] cycloadditionconditions used in these methods are analogous to those described abovefor the methods of labeling nucleic acid polymers in cells and caneasily be determined/optimized by one skilled in the art.

A. Organisms

Methods of labeling of the present invention may be performed using anyliving system that has or can develop the ability to act or functionindependently. Thus, labeling methods of the present invention may beperformed in unicellular or multicellular systems, including, humans,animals, plants, bacteria, protozoa, and fungi. In certain preferredembodiments, the labeling methods of the present invention are performedin a human or another mammal (e.g., mouse, rat, rabbit, dog, cat,cattle, swine, sheep, horse or primate).

B. Incorporation of Nucleoside Analogues by DNA Replication in LivingSystems

Administration of a nucleoside analogue to an organism may be performedusing any suitable method that results in incorporation of thenucleoside analogue into the DNA of cells of the organism.

For example, the nucleoside analogue may be formulated in accordancewith conventional methods in the art using a physiologically andclinically acceptable solution. Proper solution is dependent upon theroute of administration chosen. Suitable routes of administration can,for example, include oral, rectal, transmucosal, transcutaneous, orintestinal administration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections. Alternatively, the nucleoside analoguepreparation can be administered in a local rather than systemic manner,for example, via injection directly into a specific tissue, often in adepot or sustained release formulation.

C. [3+2] Cycloaddition

Following incorporation of the nucleotide analogue into the DNA of cellsof the organism, the step of contacting at least one cell of theorganism with a reagent comprising the second reactive unsaturated groupattached to a label may be performed by any suitable method that allowsfor the [3+2] cycloaddition to take place.

In certain embodiments, cells are collected (e.g., by drawing blood fromthe organism), isolated from a tissue obtained by biopsy (e.g., needlebiopsy, laser capture micro dissection or incisional biopsy) or isolatedfrom an organ or part of an organ (e.g., harvested at autopsy). Thecells can then be submitted to the [3+2] cycloaddition staining asdescribed above.

In other embodiments, a tissue obtained by biopsy or an organ or part ofan organ harvested at autopsy may be prepared for staining as known inthe art (e.g., fixed, embedded in paraffin and sectioned) and incubatedin the presence of the [3+2] cycloaddition reagent (e.g., afterde-waxing).

Example 4 describes an experiment carried out by the present Applicantswhere mice were intraperitoneally injected with EdU and their organsharvested 3 days after injection, prepared for staining and stained withXrhodamine-azide and with Hoechst (a fluorescent dye specific for DNAstaining). Fluorescence images of the intestine and of the brain ofthese mice are presented on FIGS. 8 and 9, and FIG. 10, respectively.

7. Isolated Labeled Nucleic Acid Polymers

In another aspect, the present invention provides isolated detectablenucleic acid polymers, for example prepared by one of the methodsdescribed herein. More specifically, the present invention providesnucleic acid polymers that are detectable following a [3+2]cycloaddition reaction as well as isolated nucleic acid polymers thatcontain at least one detectable moiety that has been incorporated via a[3+2] cycloaddition.

In certain embodiments, an inventive nucleic acid polymer contains atleast one nucleotide analogue comprising a reactive unsaturated group.Preferably, the reactive unsaturated group can undergo a [3+2]cycloaddition in the presence of a reagent comprising a differentreactive unsaturated group attached to a label.

In other embodiments, an inventive nucleic acid polymer contains atleast one nucleotide analogue attached to a label. For example, thenucleotide analogue may comprise a cycloadduct resulting from a [3+2]cycloaddition.

In still other embodiments, an inventive nucleic acid polymer containsat least one first nucleotide analogue comprising a first unsaturatedgroup and at least one second nucleotide analogue comprising a secondreactive unsaturated group. The first reactive unsaturated group cangenerally undergo a [3+2] cycloaddition in the presence of a firstreagent comprising a third reactive unsaturated group attached to afirst label, and the second reactive unsaturated group can generallyundergo a [3+2] cycloaddition in the presence of a second reagentcomprising a fourth reactive unsaturated group attached to a secondlabel.

In yet other embodiments, an inventive nucleic acid polymer contains atleast one first nucleotide analogue attached to a first label and atleast one second nucleotide analogue attached to a second label.Preferably, the first nucleotide analogue comprises a cycloadductresulting from a first [3+2] cycloaddition, and the second nucleotideanalogue comprises a cycloadduct resulting from a second [3+2]cycloaddition.

The detectable nucleic acid polymers of the present invention may beprepared by any suitable method, as described herein, includingsynthetic methods and enzymatic methods (e.g., by reverse transcriptionof total RNA).

As can be appreciated by one of ordinary skill in the art, the isolateddetectable nucleic acid polymers of the present invention may be used ina wide variety of applications.

For example, they may be used as detection probes in hybridizationassays, including microarray-based hybridization assays. In suchapplications, the detectable nucleic acid polymers are preferablyoligonucleotides (i.e., short stretches of nucleic acid sequences).Oligonucleotides used in hybridization assays generally comprise betweenabout 5 and about 150 nucleotides, for example between about 15 andabout 100 nucleotides or between about 15 and about 50 nucleotides. Thedetectable nucleic acid polymers provided by the present invention maybe used to contact an array or microarray in a hybridization assay. Insuch embodiments, the detectable nucleic acid probes may be providedwith appropriate staining reagents. Alternatively, the detectablenucleic acid polymers are provided attached to an array or micro-arrayfor a hybridization assay.

Arrays according to the present invention comprise a plurality ofdetectable nucleic acid polymers immobilized to discrete spots on asubstrate surface. Substrate surfaces can be made of any of rigid,semi-rigid or flexible materials that allow for direct or indirectattachment (i.e., immobilization) of detectable nucleic acid polymers tothe substrate surface. Suitable materials include, but are not limitedto, cellulose, cellulose acetate, nitrocellulose, glass, quartz othercrystalline substrates such as silicones, and various plastics andplastic copolymers. When fluorescence is to be detected, arrayscomprising cyclo-olefin polymers may preferably be used.

The presence of reactive functional chemical groups on the materials canbe exploited to directly or indirectly attach the detectable nucleicacid polymers to the substrate surface. Methods for immobilizingoligonucleotides to substrate surfaces to form an array are well-knownin the art.

8. Cells Comprising Detectable Nucleic Acid Polymers

In another aspect, the present invention provides cells comprisingdetectable nucleic acid polymers, for example prepared by one or more ofthe methods described herein. More specifically, the present inventionprovides cells comprising nucleic acid polymers that are detectablefollowing a [3+2] cycloaddition reaction as well as cells comprisingnucleic acid polymers that contain at least one detectable moiety thathas been incorporated via a [3+2] cycloaddition.

As will be recognized by one of ordinary skill in the art, a cell of theinvention may comprise any of the detectable nucleic acid polymersdescribed herein.

II. Determination of DNA Replication/Cellular Proliferation

In another aspect, the present invention provides methods for measuringcell proliferation and/or cell proliferation rates in a cell or anorganism.

Such methods may comprise steps of: contacting a cell with an effectiveamount of a nucleoside analogue that comprises a first reactiveunsaturated group such that the nucleoside analogue is incorporated intoDNA of the cell; contacting the cell with a reagent comprising a secondreactive unsaturated group attached to a label, such that a [3+2]cycloaddition occurs between the first and second reactive unsaturatedgroups; and determining an amount of label incorporated into the DNA tomeasure cellular proliferation. In certain embodiments, the amount oflabel gives information about the extent of cellular proliferation. Inother embodiments, the amount of label gives information about the rateof cellular proliferation.

Other methods comprise steps of: administering to an organism aneffective amount of a nucleoside analogue that comprises a firstreactive unsaturated group such that the nucleoside analogue isincorporated into DNA of cells of the organism; contacting at least onecell of the organism with a reagent comprising a second reactiveunsaturated group attached to a label, such that a [3+2] cycloadditionoccurs between the first and second reactive unsaturated groups; anddetermining an amount of label incorporated into the DNA to measurecellular proliferation in the organism. In certain embodiments, theamount of label gives information about the extent of cellularproliferation in the organism. In other embodiments, the amount of labelgives information about the rate of cellular proliferation in theorganism.

These methods may be performed using techniques and procedures asdescribed herein for methods of labeling nucleic acid polymers in cellsand organisms. With such methods, the manner of performing thecontacting and/or administering steps, type of staining reagent (i.e.,with or without a Cu chelating moiety), type of label, and techniquesfor the detection of such labels are analogous to those described forother methods of the invention relating to labeling nucleic acidpolymers in cells or in organisms.

Methods for measuring cellular proliferation or cellular proliferationrates according to the present invention may be used in a wide varietyof applications, including, but not limited to characterization of celllines, optimization of cell culture conditions, characterization ofcellular proliferation in normal, diseased and injured tissues, anddiagnosis of a variety of diseases and disorders in which cellularproliferation is involved.

A large number of diseases and disorders are known to be characterizedby altered cellular proliferation rates and thus can be monitored bymethods of the present invention. Such diseases and disorders include,but are not limited to, malignant tumors of any type (e.g., breast,lung, colon, skin, lymphoma, leukemia, and the like); pre-cancerousconditions (e.g., adenomas, polyps, prostatic hypertrophy, ulcerativecolitis, and the like); immune disorders such as AIDS, autoimmunedisorders, and primary immunodeficiencies; hematologic conditions suchas white blood cell deficiencies (e.g., granulocytopenia), anemias ofany type, myeloproliferative disorders, lymphoproliferative disordersand the like; organ failure such as alcoholic and viral hepatitis,diabetic nephropathy, myotrophic conditions, premature gonadal failureand the like; conditions affecting bones and muscles, such asosteoporosis; endocrine conditions such as diabetes, hypothyroidism andhyperthyroidism, polycystic ovaries and the like; infectious diseases,such as tuberculosis, bacterial infections, abscesses and otherlocalized tissue infections, viral infections and the like; and vasculardisorders, such as atherogenesis, cardiomyopathies, and the like.

III. Methods of Use

Methods for labeling nucleic acid polymers and for measuring cellularproliferation or cellular proliferate rates as disclosed herein, can beused in a wide variety of applications, some of which are describedbelow.

1. Screening Assays

In another aspect, the present invention provides methods for theidentification of agents that perturb cellular proliferation. Thesemethods may be used for screening agents for their ability to induce(i.e., increase, enhance or otherwise exacerbate) or inhibit (i.e.,decrease, slow down or otherwise suppress) cell proliferation.

For example, such methods may comprise steps of: (a) contacting a cellwith a test agent; (b) contacting the cell with an effective amount of anucleoside analogue that comprises a first reactive unsaturated groupsuch that the nucleoside analogue is incorporated into DNA of the cell;(c) contacting the cell with a reagent comprising a second reactiveunsaturated group attached to a label, such that a [3+2] cycloadditionoccurs between the first and second reactive unsaturated groups; (d)determining an amount of label incorporated into the DNA, wherein theamount of label indicates the extent or rate of cellular proliferation;and (e) identifying the test agent as an agent that perturbs cellularproliferation if the amount of label measured in step (d) is less thanor greater than the amount of label measured in a control application inwhich the cell is not contacted with the test agent.

In certain embodiments, the cell is contacted with the test agent afterit is contacted with the nucleoside analogue (i.e., step (b) isperformed before step (a)).

The manner of performing the steps of contacting the cell; the stainingreagent; the label type; and methods of detecting the labeled nucleicacid polymers are analogous to those described for other methods of theinvention relating to measuring cellular proliferation and cellularproliferation rates in cells in vitro.

As will be appreciated by one of ordinary skill in the art, thescreening methods of the present invention may also be used to identifycompounds or agents that regulate cellular proliferation (i.e.,compounds or agents that can decrease, slow down or suppressproliferation of over-proliferative cells or that can increase, enhanceor exacerbate proliferation of under-proliferative cells).

A. Cells for Screening Assays

The screening assays of the present invention may be performed using anynormal or transformed cells that can be grown in standard tissue cultureplastic ware. Cells may be primary cells, secondary cells, orimmortalized cells. Preferably, cells to be used in the inventivescreening methods are of mammalian (human or animal) origin. Cells maybe from any organ or tissue origin and of any cell types, as describedabove.

Selection of a particular cell type and/or cell line to perform ascreening assay according to the present invention will be governed byseveral factors such as the nature of the agent to be tested and theintended purpose of the assay. For example, a toxicity assay developedfor primary drug screening (i.e., first round(s) of screening) maypreferably be performed using established cell lines, which arecommercially available and usually relatively easy to grow, while atoxicity assay to be used later in the drug development process maypreferably be performed using primary or secondary cells, which areoften more difficult to obtain, maintain, and/or grow than immortalizedcells but which represent better experimental models for in vivosituations.

In certain embodiments, the screening methods are performed using cellscontained in a plurality of wells of a multi-well assay plate. Suchassay plates are commercially available, for example, from StrategeneCorp. (La Jolla, Calif.) and Corning Inc. (Acton, Mass.), and include,for example, 48-well, 96-well, 384-well and 1536-well plates.

B. Test Agents

As will be appreciated by those of ordinary skill in the art, any kindof compounds or agents can be tested using the inventive methods. A testcompound may be a synthetic or natural compound; it may be a singlemolecule, a mixture of different molecules or a complex of differentmolecules. In certain embodiments, the inventive methods are used fortesting one or more compounds. In other embodiments, the inventivemethods are used for screening collections or libraries of compounds.

Compounds that can be tested for their capacity or ability to perturb(i.e., induce or inhibit) or regulate cell proliferation can belong toany of a variety of classes of molecules including, but not limited to,small molecules, peptides, saccharides, steroids, antibodies (includingfragments or variants thereof), fusion proteins, antisensepolynucleotides, ribozymes, small interfering RNAs, peptidomimetics, andthe like.

Compounds or agents to be tested according to methods of the presentinvention may be known or suspected to perturb or regulate cellproliferation. Alternatively, the assays may be performed usingcompounds or agents whose effects on cell proliferation are unknown.

Examples of compounds that may affect cell proliferation and that can betested by the methods of the present invention include, but are notlimited to, carcinogens; toxic agents; chemical compounds such assolvents; mutagenic agents; pharmaceuticals; particulates, gases andnoxious compounds in smoke (including smoke from cigarette, cigar andindustrial processes); food additives; biochemical materials; hormones;pesticides; ground-water toxins; and environmental pollutants. Examplesof agents that may affect cell proliferation and that can be tested bythe methods of the present invention include, but are not limited to,microwave radiation, electromagnetic radiation, radioactive radiation,ionizing radiation, heat, and other hazardous conditions produced by orpresent in industrial or occupational environments.

C. Identification of Agents that Induce or Inhibit CellularProliferation

According to screening methods of the present invention, determinationof the ability of a test agent to perturb or regulate cellularproliferation includes comparison of the amount of label incorporatedinto DNA of a cell that has been contacted with the test agent with theamount of label incorporated into DNA of a cell that has not beencontacted with the test agent.

A test agent is identified as an agent that perturbs cellularproliferation if the amount of label incorporated into DNA of the cellthat has been contacted with the test agent is less than or greater thanthe amount of label measured in the control cell. More specifically, ifthe amount of label incorporated into DNA of the cell that has beencontacted with the test agent is less than the amount of label measuredin the control cell, the test agent is identified as an agent thatinhibits cell proliferation. If the amount of label incorporated intoDNA of the cell that has been contacted with the test agent is greaterthan the amount of label measured in the control cell, the test agent isidentified as an agent that induces cell proliferation.

Reproducibility of the results may be tested by performing the analysismore than once with the same concentration of the test agent (forexample, by incubating cells in more than one well of an assay plate).Additionally, since a test agent may be effective at varyingconcentrations depending on the nature of the agent and the nature of itmechanism(s) of action, varying concentrations of the test agent may betested (for example, added to different wells containing cells).Generally, test agent concentrations from 1 fM to about 10 mM are usedfor screening. Preferred screening concentrations are between about 10pM and about 100 μM.

In certain embodiments, the methods of the invention further involve theuse of one or more negative or positive control compounds. A positivecontrol compound may be any molecule or agent that is known to perturb(i.e., induce or inhibit) or regulate cellular proliferation. A negativecontrol compound may be any molecule or agent that is known to have nodetectable effects on cellular proliferation. In these embodiments, theinventive methods further comprise comparing the effects of the testagent to the effects (or absence thereof) of the positive or negativecontrol compound.

As will be appreciated by those skilled in the art, it is generallydesirable to further characterize an agent identified by the inventivescreening methods as an agent that perturbs or an agent that regulatescellular proliferation. For example, if a test compound has beenidentified as an agent that perturbs (or regulates) cellularproliferation using a given cell culture system (e.g., an establishedcell line), it may be desirable to test this ability in a different cellculture system (e.g., primary or secondary cells).

Test agents identified by the screening methods of the invention mayalso be further tested in assays that allow for the determination of theagents' properties in vivo.

Accordingly, the present invention provides methods for identifying anagent that perturbs cellular proliferation or cell proliferation rate invivo. Such methods comprise steps of: (a) exposing an organism to a testagent; (b) administering to the organism an effective amount of anucleoside analogue that comprises a first reactive unsaturated groupsuch that the nucleoside analogue is incorporated into DNA of cells ofthe organism; (c) contacting at least one cell of the organism with areagent comprising a second reactive unsaturated group attached to alabel, such that a [3+2] cycloaddition occurs between the first andsecond reactive unsaturated group; (d) determining an amount of labelincorporated into the DNA, wherein the amount of label indicates theextent of cellular proliferation or rate of cellular proliferation; and(e) identifying the test agent as an agent that perturbs cellularproliferation in the organism if the amount of label measured in step(d) is less than or greater than the amount of label measured in acontrol application in which the organism is not exposed to the testagent.

In certain embodiments, the test agent is administered after theorganism has been contacted with the nucleoside analogue (i.e., step (b)is performed prior to step (a)).

As will be appreciated by one of ordinary skill in the art, thesemethods can be used to identify agents that regulate cellularproliferation in vivo.

The manner of administration, staining reagent, type of label and methodof detection of the labeled nucleic acid polymers are analogous to thosedescribed herein for other methods of the invention relating tomeasuring cellular proliferation in living systems.

2. Cell Cycle Studies

The labeling methods of the present invention, which do not requirefixation and denaturation and are therefore suitable for application inliving cells, can be used in studies of the complex spatio-temporalmechanisms of the cell cycle. A clear understanding of the mechanism ofcell cycle in the presence or absence of various perturbations can pavethe way to the development of new therapeutic approaches for controllingor treating human diseases, such as cancer. Until recently, most studiesof nuclear architecture were carried out in fixed cells (A. I. Lamondand W. C. Earnshaw, Science, 1998, 280: 457-553). However, time-lapsefluorescence microscopy imaging has since been demonstrated to allowlive cell nuclei to be observed and studied in a dynamic fashion, and toprovide far richer information content than conventional fixed cellmicroscopy techniques (Y. Hiraoka and T. Haraguchi, Chromosome Res.,1996, 4: 173-176; T. Kanda et al., Curr. Biol., 1998, 8: 377-385).

As shown on FIG. 11, the methods of the present invention allow specificlabeling of DNA undergoing replication as well as characterization of acell as mitotic (e.g., anaphase) or interphase. Thus, combining thelabeling methods disclosed herein and imaging techniques such astime-lapse fluorescence microscopy or flow cytometry can help acquirefundamental knowledge about the cell cycle of different cell types undervarious perturbation conditions and allow the development of new drugsthat affect the cell cycle.

Thus, the methods of the present invention may be applied to the studyof a large variety of pathological conditions. For example, cancer isincreasingly viewed as a cell cycle disease. This view reflects theevidence that the vast majority of tumors have suffered defects thatderail the cell cycle machinery leading to increased cell proliferation.Such defects can target either components of the cell cycle itself orelements of upstream signaling cascades that eventually converge totrigger cell cycle events. Cancer is not the only clinical conditionthought to be associated with cell cycle deregulation (M. D. Garrett,Curr. Sci. 2001, 81: 515-522). For example, the mechanism by whichneurons die in human neurodegenerative diseases remains an enigma tilltoday (I. Vincent et al., Prog. Cell Cycle Res., 2003, 5: 31-41).Terminally differentiated neurons of normal brains are incapable of celldivision. However, accumulating evidence has suggested that aberrantactivation of the cell cycle in certain neurodegenerative diseases leadsto their demise. Elucidating the details of this cell cycle-mediateddegenerative cascade may lead to novel strategies for curbing the onsetand progression of certain neurodegenerative diseases. Similarly, it isknown that manipulation of cell division can have beneficial orpathological consequences on cardiovascular function (M. Boehm and E. G.Nabel, Prog. Cell Cycle Res., 2003, 5: 19-30). The inability ofcardiomyocytes to proliferate and regenerate following injury results inan impairment of cardiac function associated with physical impedimentand may lead to death. The genetic program in the cardiomyocytes thatleads to their inability to proliferate and regenerate is notunderstood, but if identified, it could lead to therapies aimed atre-initiating the cell cycle and proliferation in cardiomyotes.

3. Chromosomal Structures

The labeling methods of the present invention can also be used for thestudy of chromosomes' ultrastructures.

For example, the labeling methods of the present invention may be usedto study sister chromatic exchange (SCE). SCE is a natural process inwhich two sister chromatids break and rejoin with one another physicallyswitching positions on the chromosome (S. Wolf, Annu. Rev. Genet., 1977,11: 183-201; S. A. Latt, Annu. Rev. Genet., 1981, 15: 11-53). Suchexchanges take place during cell replication with about 10 SCEsoccurring spontaneously in normally cycling human cells (P. E Crossen etal., Hum. Genet., 1977, 35: 345-352; S. M. Galloway and H. J. Evans,1975, 15: 17-29). SCEs can also be induced by various genotoxictreatments (L. Hagmar et al., Cancer Res., 1998, 58: 4117-4121)suggesting that SCEs reflect a DNA repair process. Detection of SCEsrequires some means of differentially labeling sister chromatids andthat has traditionally been done by growing cells in a medium containingBrdU for the duration of two complete cell cycles. The labeling methodsof the present invention may be used instead of BrdU (see FIG. 12). Asshown on FIG. 13, the labeling methods disclosed herein allow stainingof only one DNA molecule of the two that form a chromosome.

The labeling methods of the present invention may also be used as a newtool to study kinetochore-microtubule interactions and centromericcohesion. Proper segregation of chromosomes during cell division isessential for the maintenance of genetic stability. During this processchromosomes must establish stable functional interactions withmicrotubules through the kinetochore, a specialized protein structurelocated on the surface of the centromeric heterochromatin. Stableattachment of kinetochores to a number of microtubules results in theformation of a kinetochore fiber that mediates chromosome movement.Although the fidelity of chromosome segregation depends on preciseinteractions between kinetochores and microtubules, it is still unclearhow this interaction is mediated and regulated (M. B. Gordon et al., J.Cell Biol., 2001, 152: 425-434; A. A. Van Hooser and R. Heald, Curr.Biol., 2001, 11: R855-857; S. Biggins and C. E. Walczak, Curr. Biol.,2003, 13: R449-460; H. Maiato et al., J. Cell Sci., 2004, 117:5461-5477; H. Maiato and C. E. Sunkel, Chromosome Res., 2004, 12:585-597) The centromere is a specialized region of the chromosome thatis essential for proper segregation of the chromosomes during celldivision. It is the site at which the kinetochore is assembled. Duringmitosis, replicated sister chromatids must maintain cohesion as theyattach to the mitotic spindle. At anaphase, cohesion is lostsimultaneously along the entire chromosome, releasing sisters from oneanother and allowing them to segregate to opposite poles. The molecularmechanism(s) responsible for chromatic cohesion during mitosis remain(s)ambiguous (K. J. Dej and T. L. Orr-Weaver, Trends Cell Biol., 2000, 10:392-399; T. Fukagawa, Chromosome Res., 2004, 12: 557-567; S. Salic etal., Cell, 2004, 118: 567-578).

4. Labeling of RNA and RNA Localization Studies

The labeling methods of the present invention may be used for labelingRNA. The inventive methods include a [3+2] cycloaddition between a firstreactive unsaturated group on a nucleotide analogue incorporated into aribonucleotide polymer and a second reactive unsaturated group attachedto a label. The [3+2] cycloaddition reaction results in labeling of theribonucleotide polymer.

In such methods, the ribonucleotide polymer comprising the nucleotideanalogue may be prepared by any suitable method, as known in the art.For example, the ribonucleotide polymer may be synthesized by in vitrotranscription of DNA, cloned downstream of T3, T7 or SP6 polymerasespromoters in the presence of nucleotide triphosphates (including thenucleotide analogue triphosphate) as substrates. Alternatively, theribonucleotide polymer may be prepared using amplification methods.

The inventive labeling methods may be used in microarray hybridizationassays to measure mRNA transcript levels of many genes in parallel.

The inventive labeling methods may also find applications in ribosomedisplay, a cell-free system for the in vitro selection of proteins andpeptides (C. Tuerk and L. Gold, Science 1990, 249: 505-510; G. F. Joyce,Gene 1989, 82: 83-87; J. W. Szostak, Trends Biochem. Sci. 1992, 17:89-93; D. E. Tsai et al., Proc. Natl. Acad. Sci. USA, 1992, 89:8864-8868; J. A. Doudna et al., Proc. Natl. Acad. Sci. USA, 1995, 92:2355-2359; C. Shaffitzel et al., J. Immunol. Methods, 1999, 231:119-135; D. Lipovsel and A. Pluckthun, J. Immunol. Methods, 2001, 290:51-67; A. M. Jackson et al., Brief Funct. Genomic Protreomic, 2004, 2:308-319). These selection assays generally involve adding an RNA libraryto the protein or molecule of interest, washing to remove unbound RNA,and specifically eluting the RNA bound to the protein. The RNA is thenreversed-transcribed and amplified by PCR. The cDNA obtained is thentranscribed in the presence of nucleotide analogues for detectionpurposes. Those molecules that are found to bind the protein or othermolecule of interest are cloned and sequenced to look for commonsequences. The common sequence is then used to develop therapeuticoligonucleotides.

The RNA labeling methods of the present invention may also be used forvisualizing mRNA movement (transport and localization) in living cells.mRNA localization is a common mode of post-transcriptional regulation ofgene expression that targets a protein to its site of function (I. M.Palacios and D. St Johnston, Annu. Rev. Cell Dev. Biol., 2001, 17:569-614; R. P. Jansen, Nature Rev. Mol. Cell. Biol., 2001, 2: 247-256;M. Kloc et al., Cell, 2002, 108: 533-544). Many of the bestcharacterized localized mRNAs are found in oocytes and early embryos,where they function as localized determinants that control axisformation and the development of the germline. mRNA localization hasalso been shown to play an important role in somatic cells, such asneurons, where it may be involved in learning and memory. Different mRNAvisualization methods have been developed to identify the machinery andmechanisms involved in mRNA transport and localization, includingaminoally-uridine triphosphate incorporation into RNA followed byfluorescein or rhodamine coupling and direct incorporation ofAlexa-Fluor-uridine triphosphate into RNA. (V. Van de Bor and I. Davis,Curr. Opin. Cell Biol., 2004, 16: 300-307). mRNA molecules fluorescentlylabeled in vitro according to the present invention may be introducedinto living cells and their movement monitored in real time.

IV. Kits

In another aspect, the present invention provides kits comprisingmaterials useful for carrying out one or more of the methods of theinvention. The inventive kits may be used by diagnostic laboratories,clinical laboratories, experimental laboratories, or practitioners. Theinvention provides kits which can be used in these different settings.

Basic materials and reagents for labeling nucleic acid polymersaccording to the present invention may be assembled together in a kit.An inventive kit for labeling a nucleic acid polymer may include atleast one nucleoside analogue that comprises a first reactiveunsaturated group; and a reagent comprising a second reactiveunsaturated group attached to a label. An inventive kit for duallabeling of a nucleic acid polymer may include at least one firstnucleoside analogue that comprises a first reactive unsaturated group;at least one second nucleoside analogue that comprises a second reactiveunsaturated group; a first reagent comprising a third reactiveunsaturated group attached to a first label; and a second reagentcomprising a fourth reactive unsaturated group attached to a secondlabel. Each kit preferably comprises the reagents which render theprocedure specific. Thus, if the detectable agent is a hapten, the kitwill preferably comprise the corresponding appropriate antibody.Similarly, a kit intended to be used for the labeling of nucleic acidpolymers in living organisms will contain nucleosides formulated suchthat they can be administered to a living organism. A kit intended to beused for screening compounds for their ability to induce or inhibitcellular proliferation may include cells comprising labeled nucleic acidpolymers of the present invention.

Certain inventive kits may further comprise buffers and/or reagentsuseful to perform a [3+2] cycloaddition reaction, such as aqueous mediumand Cu(I).

An inventive kit may further comprise one or more of: wash buffersand/or reagents, cell fixation buffers and/or reagents,immunohistochemical buffers and/or reagents, DAB photoconversion buffersand/or reagents, and detection means. The buffers and/or reagents arepreferably optimized for the particular labeling/detection technique forwhich the kit is intended. Protocols for using these buffers andreagents for performing different steps of the procedure may also beincluded in the kit.

Kits may also contain instruments (e.g., needle biopsy syringe) and/orreagents for the isolation of cells from an organism.

The reagents may be supplied in a solid (e.g., lyophilized) or liquidform. The kits of the present invention optionally comprise differentcontainers (e.g., vial, ampoule, test tube, flask or bottle) for eachindividual buffer and/or reagent. Each component will generally besuitable as aliquoted in its respective container or provided in aconcentrated form. Other containers suitable for conducting certainsteps of the labeling/detection assay may also be provided. Theindividual containers of the kit are preferably maintained in closeconfinement for commercial use.

Instructions for using the kit according to one or more methods of theinvention may comprise instructions for labeling nucleic acid polymers,instructions for measuring cellular proliferation, instructions forinterpreting the results obtained as well as a notice in the formprescribed by a governmental agency (e.g., FDA) regulating themanufacture, use or sale of pharmaceuticals or biological products.

EXAMPLES

The following examples describe some of the preferred modes of makingand practicing the present invention. However, it should be understoodthat these examples are for illustrative purposes only and are not meantto limit the scope of the invention. Furthermore, unless the descriptionin an Example is presented in the past tense, the text, like the rest ofthe specification, is not intended to suggest that experiments wereactually performed or data were actually obtained.

Example 1 General Protocols for Labeling of Cells with Ethynyl-dU

Ethynyl-dU (EdU) was used in tissue culture media (DMEM complementedwith penicillin, streptomycin, and fetal calf serum (FCS)) ranging from10 nM to 1 μM depending on the length of the labeling pulse. Forexample, if cells to be labeled were synchronized in the S phase, 100 μMto 1 μM of EdU was used for 1 to 2 hours. After labeling, the cells werewashed 3 or 4 times with PBS and then tissue culture media was added.

Staining of Living Cells:

Staining Solution: 100 mM Tris pH 8.5 (from 2 M stock in water), 0.5 to1 mM CuSO₄ (from 1 M stock in water), 0.5 to 1 μM TMR-propyl-azide (fromapproximately 100 mM stock in DMF), and water (as required) were mixedtogether. Ascorbic acid (from a 0.5 mM stock in water) was then added tothis solution to a final concentration of 50 mM, and the resultingstaining solution is mixed thoroughly.

For staining cells alive without permeabilization (e.g., when thestaining reagent was TMR-propyl-azide, which is cell permeable), tissueculture media was removed and replaced by the staining solutiondescribed above. Cells were incubated for at least 30 minutes in thepresence of the staining solution although staining is generallycomplete after 10 minutes of incubation. After staining, unreactedTMR-propyl-azide was removed by washing with buffer (such as PBS or TBS)containing 0.5% Triton-X100 (or similar detergent). Washes with methanolor ethanol may be performed to obtain low background

If desired, cell fixation can be performed at the same time by adding 3%formaldehyde (or glutaraldehyde) to the washing buffer. In this case,cells were incubated in the washing buffer for at least 10 minutes atroom temperature.

Staining of Fixed Cells:

Staining Solution: 100 mM Tris pH 8.5 (from 2 M stock in water), 0.5 to1 mM CuSO₄ (from 1 M stock in water), 0.5 to 1 μM fluorophore-azide(from approximately 10-100 mM stock in DMSO), and water (as required)were mixed together. Ascorbic acid (from a 0.5 mM stock in water) wasthen added to this solution to a final concentration of 50-100 mM, andthe resulting staining solution is mixed thoroughly using a vortex.

Cells can be fixed by any suitable method, e.g., cells can be fixed byaldehyde fixation (formaldehyde or glutaraldehyde), with or withoutpermeabilization. After fixation, the cells were washed in buffer withor without non-ionic detergent (0.2-0.5% Triton X100). Before staining,cells were rinsed with buffer without detergent (PBS or TBS). The cellswere then incubated in the presence of the staining solution for atleast 30 minutes. Overnight staining is preferably performed in a coldroom.

Following staining, the cells were washed several times with a buffercontaining 0.5% of detergent. Washes with methanol or ethanol can beperformed to significantly reduce the background if necessary.

Stained Cells Imaging:

Stained cells can then be immunostained using standard protocols. Cellswere then mounted in standard mounting media and imaged. Whether mountedor not, the stain was found to be very stable indefinitely at 4° C.

Example 2 Labeling of HeLa Cells with Ethynyl-dU

HeLa cells were labeled or not with 1 μM EdU as described in Example 1.They were then fixed/permeabilized and stained with an Xrhodamine-azidereagent (which is not cell permeable and therefore requirespermeabilization in order to perform the click chemistry).

Images of both populations of cells (i.e., labeled or not with EdU) arepresented on FIG. 5. A higher resolution view of these cells ispresented in FIG. 11 (right), along with images obtained fromEdU-labeled HeLa cells stained with OliGreen (FIG. 11 left). Note thespeckled appearance of the EdU stain in the bottom cell which isundergoing anaphase. The speckles are due to the fact that the labelingpulse of EdU was shorter than the time required for the cell toreplicate its DNA; thus only the DNA replicating during the pulse waslabeled.

Example 3 Time-Lapse Fluorescence Imaging of Labeled Live Cells

To stain DNA under conditions as close to the native state as possible,a cell-permeable TMR-azide reagent was developed. TMR-propyl-azide wassynthesized by first reacting TMR-carboxy-NHS ester with3-bromo-propylamine to form bromo-propyl-TMR. The latter compound wasthen reacted with sodium azide to form TMR-propyl-azide, which was foundto be cell-permeable and thus suitable for labeling cells without theneed for permeabilization and/or fixation.

To demonstrate staining of cells using TMR-propyl-azide, HeLa cells werefirst labeled with EdU. To facilitate live microscopic imaging,EdU-labeled cells were plated in coverslip chambers which were mountedon the heated stage (37° C.) of an inverted Nikon TE200)U microscopeequipped for wide-field fluorescence microscopy as well as spinning-diskconfocal fluorescence microscopy (Yokogama spinning disk confocal headfrom Perkin-Elmer). The media covering the cells was removed andreplaced with a staining solution containing 200 nM TMR-propyl-azide, 25mM ascorbate and 1 mM copper (II) sulfate dissolved in physiologicalsaline buffer. The cells were imaged by time-lapse fluorescentmicroscopy (one frame every 15 seconds) to detect the TMR signalaccumulating in the nuclei of the EdU-labeled cells.

A shown on FIG. 6 (which presents images obtained by time-lapsefluorescence microscopy), the click reaction was found to work very wellon live cells using this cell permeable reagent. As shown on FIG. 7, EdUdetection was complete within minutes.

Example 4 In Vivo Labeling

BLAB/C mice were injected with 200 micrograms of EdU intraperitoneally.Three days later, organs were harvested, fixed, embedded in paraffin andsectioned. The sections were then de-waxed and stained withXrhadomine-azide for 5 minutes, stained with Hoechst, washed and thenmounted.

FIG. 8 shows a high magnification (400×) of a section through theintestine. The cells with red nuclei are the cells which incorporatedEdU and their descendants. DNA appears in blue. FIG. 9 is a composite ofimage of many 400× images stitched together to form the image of a largepiece of a mouse intestine section (DNA appears in green). The object isan entire oblique section through the intestine—about half a centimeterin length.

FIG. 10 is a section through the mouse brain, an organ whose cellsalmost never divide (unlike the intestine, which is highlyproliferative). A sole EdU-labeled cell can be easily identified on thisbrain section.

Example 5 Tracing a Single Sister Chromatic at the Centromere

The purpose of the experiment reported herein was to label only one DNAmolecule of the two that form a chromosome. That was accomplished bypulsing HeLa cells with EdU during DNA synthesis followed by a chaselasting two cell cycles, as depicted on FIG. 12.

In the first mitosis after labeling, both DNA molecules were labeledwhile in the second mitosis only one DNA molecule was labeled. EdUallowed very nice resolution imaging of chromosomes in cells as shown byFIG. 13. In these experiments, Alexa568-azide was used for the stainingand the staining process involved permeabilization.

Example 6 Labeling of RNA in Cells

EU (ethynyl-uridine) was synthesized as a label for cellular RNA. HeLacells were labeled with 10 μM EU overnight, fixed and stained withXrhodamine-azide. As shown on FIG. 14, cells that were stained withXrhodamine-azide without having been labeled with EU (negative controlshowed very little staining, as expected. EU-labeled cells stained withXrhodamine-azide exhibited strong cytoplasmic staining indicative of theEU having been incorporated into cellular RNA.

Example 7 Using Azido-dU (AdU) to Stain HeLa Cells

The 5-azido derivatives of both 2′-deoxyuridine (AdU) and uridine (AU)were synthesized.

AdU is a deoxynucleoside analogue that can be used together with EdU tostain DNA with two different colors (if EdU and AdU are administered tocells as two separate pulses and then the detection is done usingfluorophore 1-azide and fluorophore 2-terminal alkyne, respectively).

Similarly, AU can be used to label cellular RNA. Cellular RNA can belabeled with two different colors using ethynyl-uridine andazido-uridine, respectively.

Here HeLa cells were labeled overnight in 10 μM AdU and then stainedwith 10 μM Alexa568-alkyne using the conditions described herein forstaining cells labeled with EdU. FIG. 16 presents two pictures of afield of the stained HeLa cells. As shown on FIG. 16, AdU is clearlydetected in the nuclei of the labeled interphase cells as well as in thecondensed chromosomes of mitotic cells.

Example 8 Erasing DNA Staining Under Mild Conditions

In some cases, it is desirable to erase the fluorescent staining ofEdU-labeled DNA. A strategy is described here to accomplish that goal.

HeLa cells were labeled with 1 μM EdU overnight and fixed the next day.Staining was performed using 10 μM of an Alexa568-azide (abbreviatedAlexa568-SS-azide) that was synthesized by the Applicant in which theazide group was attached to the fluorophore via a cystamine linker(H₂N—CH₂—CH₂—S—S—CH₂—CH₂—NH₂). The disulfide bond in the linker caneasily reduced using a reducing agent such as DTT (dithiothreitol),resulting in the dissociation of the fluorophore from the DNA strands.

The second row of FIG. 17 shows pictures of HeLa cells which wereincubated with 20 mM (left hand side) and 100 mM (right hand side) ofDTT, after the Alexa568-SS-azide stain, as described above. DTTincubation was carried out for 30 minutes at 37° C. The first row ofFIG. 17 shows pictures of HeLa cells incubated as described above (i.e.,without subsequent DTT incubation) (right panel) and HeLa cells thatwere not incubated in the presence of EdU and Alexa568-azide (leftpanel). As shown by the pictures of FIG. 17, DTT incubation effectivelyremoves the fluorescent signal from cell nuclei.

OTHER EMBODIMENTS

Other embodiments of the invention will be apparent to those skilled inthe art from a consideration of the specification or practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope of theinvention being indicated by the following claims.

1. A method of labeling a nucleic acid polymer, comprising steps of: providing a nucleic acid polymer comprising at least one nucleotide analogue that comprises a first reactive unsaturated group; and contacting the nucleic acid polymer with a reagent comprising a second reactive unsaturated group attached to a label, such that a [3+2] cycloaddition occurs between the first and second unsaturated groups.
 2. The method of claim 1, wherein the first reactive unsaturated group comprises a 1,3-dipole and the second reactive unsaturated group comprises a dipolarophile or wherein the first reactive unsaturated group comprises a dipolarophile and the second reactive unsaturated group comprises a 1,3-dipole.
 3. The method of claim 2, wherein the 1,3-dipole comprises an azide group and the dipolarophile comprises an ethynyl bond.
 4. The method of claim 1, wherein the label is directly detectable.
 5. The method of claim 4, wherein the label comprises a fluorescent agent.
 6. The method of claim 1, wherein the label is indirectly detectable.
 7. The method of claim 6, wherein the label comprises a hapten.
 8. The method of claim 1, wherein the nucleic acid polymer is inside a cell or in an organism.
 9. The method of claim 8, wherein the at least one nucleotide analogue is incorporated into the nucleic acid polymer during DNA replication.
 10. The method of claim 1, wherein the step of contacting is performed under aqueous conditions in presence of Cu(I).
 11. The method of claim 8, wherein the step of contacting is performed under aqueous conditions in absence of Cu(I) and the reagent further comprises a Cu chelating moiety.
 12. A method of dually labeling a nucleic acid polymer, comprising steps of: providing a nucleic acid polymer comprising at least one first nucleotide analogue that comprises a first reactive unsaturated group and at least one second nucleotide analogue that comprises a second reactive unsaturated group; contacting the nucleic acid polymer with a first reagent comprising a third reactive unsaturated group attached to a first label, such that a [3+2] cycloaddition occurs between the first and third unsaturated groups; and contacting the nucleic acid polymer with a second reagent comprising a fourth reactive unsaturated group attached to a second label, such that a [3+2] cycloaddition occurs between the second and fourth unsaturated groups.
 13. The method of claim 12, wherein the first reactive unsaturated group comprises a first 1,3-dipole and the third reactive unsaturated group comprises a first dipolarophile, and wherein the second reactive unsaturated group comprises a second dipolarophile and the fourth reactive unsaturated group comprises a second 1,3-dipole.
 14. The method of claim 12, wherein the first reactive unsaturated group comprises a first dipolarophile and the third reactive unsaturated group comprises a first 1,3-dipole, and wherein the second reactive unsaturated group comprises a second 1,3-dipole and the fourth reactive unsaturated group comprises a second dipolarophile.
 15. The method of claim 13 or 14, wherein the first 1,3-dipole comprises a first azide group and the second 1,3-dipole comprises a second azide group and wherein the first dipolarophile comprises a first ethynyl group and the second dipolarophile comprises a second ethynyl group.
 16. The method of claim 12, wherein the first and second labels are directly detectable.
 17. The method of claim 16, wherein the first label comprises a first fluorescent agent, the second label comprises a second fluorescent agent, and the first and second fluorescent agents produce a dual-color fluorescence upon excitation.
 18. The method of claim 12, wherein the first and second labels are indirectly detectable.
 19. The method of claim 18, wherein the first label comprises a first hapten and the second label comprises a second hapten.
 20. The method of claim 12, wherein the nucleic acid polymer is inside a cell or in an organism.
 21. The method of claim 20, wherein the at least one first nucleotide analogue and at least second one nucleotide analogue are incorporated into the nucleic acid polymer during DNA replication.
 22. The method of claim 12, wherein the steps of contacting are performed simultaneously.
 23. The method of claim 12, wherein the steps of contacting are performed sequentially.
 24. The method of claim 12, wherein the steps of contacting are performed under aqueous conditions in presence of Cu(I).
 25. The method of claim 20, wherein the steps of contacting are performed under aqueous conditions in absence of Cu(I) and the reagent further comprises a Cu chelating moiety.
 26. A method of differentially labeling nucleic acid polymers, comprising steps of: providing a first nucleic acid polymer comprising at least one first nucleotide analogue that comprises a first reactive unsaturated group; providing a second nucleic acid polymer comprising at least one second nucleotide analogue that comprises a second reactive unsaturated group; contacting the first nucleic acid polymer with a first reagent comprising a third reactive unsaturated group attached to a first label, such that a [3+2] cycloaddition occurs between the first and third unsaturated groups; and contacting the second nucleic acid polymer with a second reagent comprising a fourth reactive unsaturated group attached to a second label, such that a [3+2] cycloaddition occurs between the second and fourth unsaturated groups.
 27. The method of claim 26, wherein the first reactive unsaturated group comprises a first 1,3-dipole and the third reactive unsaturated group comprises a first dipolarophile, and the second reactive unsaturated group comprises a second dipolarophile and the fourth reactive unsaturated group comprises a second 1,3-dipole.
 28. The method of claim 26, wherein the first reactive unsaturated group comprises a first dipolarophile and the third reactive unsaturated group comprises a first 1,3-dipole, and the second reactive unsaturated group comprises a second 1,3-dipole and the fourth reactive unsaturated group comprises a second dipolarophile.
 29. The method of claim 27 or 28, wherein the first 1,3-dipole comprises a first azide group and the second 1,3-dipole comprises a second azide group and wherein the first dipolarophile comprises a first ethynyl group and the second dipolarophile comprises a second ethynyl group.
 30. The method of claim 26, wherein the first and second labels are directly detectable.
 31. The method of claim 30, wherein the first label comprises a first fluorescent agent, the second label comprises a second fluorescent agent, and the first and second fluorescent agents produce a dual-color fluorescence upon excitation.
 32. The method of claim 26, wherein the first and second labels are indirectly detectable.
 33. The method of claim 32, wherein the first label comprises a first hapten and the second label comprises a second hapten.
 34. The method of claim 25, wherein the first nucleic acid polymer is inside a first cell and the second nucleic acid polymer is inside a second cell.
 35. The method of claim 26, wherein the first nucleic acid polymer is inside a first organism and the second nucleic acid polymer is inside a second organism.
 36. The method of claim 34 or 35, wherein the at least one first nucleotide analogue is incorporated into the first nucleic acid polymer during DNA replication, and the at least one second nucleotide analogue is incorporated into the second nucleic acid polymer during DNA replication.
 37. The method of claim 26, wherein the steps of contacting are performed simultaneously.
 38. The method of claim 26, wherein the steps of contacting are performed sequentially.
 39. The method of claim 35, wherein the steps of contacting are performed under aqueous conditions in presence of Cu(I).
 40. The method of claim 34 or 35, wherein the steps of contacting are performed under aqueous conditions in absence of Cu(I) and the first reagent further comprises a first Cu chelating moiety and the second reagent further comprises a second Cu chelating moiety.
 41. A nucleic acid polymer comprising at least one nucleotide analogue attached to a label, wherein said nucleic acid polymer is prepared by the method of claim
 1. 42. The nucleic acid polymer of claim 41, wherein the at least one nucleotide analogue comprises a cycloadduct resulting from a [3+2] cycloaddition between an ethynyl group and an azide group.
 43. The nucleic acid polymer of claim 41, wherein the label is directly detectable.
 44. The nucleic acid polymer of claim 43, wherein the label comprises a fluorescent agent.
 45. The nucleic acid polymer of claim 41, wherein the label is indirectly detectable.
 46. The nucleic acid polymer of claim 45, wherein the label comprises a hapten.
 47. A nucleic acid polymer comprising at least one first nucleotide analogue attached to a first label and at least one second nucleotide analogue attached to a second label, wherein said nucleic acid polymer is prepared by the method of claim
 12. 48. The nucleic acid polymer of claim 47, wherein the at least one first nucleotide analogue comprises a first cycloadduct resulting from a [3+2] cycloaddition between a first ethynyl group and a first azide group and the at least one second nucleotide analogue comprises a second cycloadduct resulting from a [3+2] cycloaddition between a second ethynyl group and an second azide group.
 49. The nucleic acid polymer of claim 47, wherein the first and second labels are directly detectable.
 50. The nucleic acid polymer of claim 49, wherein the first label comprises a first fluorescent agent, the second label comprises a second fluorescent agent, and the first and second fluorescent agent produce a dual fluorescence upon excitation.
 51. The nucleic acid polymer of claim 47, wherein the first and second label are indirectly detectable.
 52. The nucleic acid polymer of claim 51, wherein the first label comprises a first hapten, and the second label comprises a second hapten.
 53. A cell comprising a nucleic acid polymer according to claim 42 or
 47. 54. A kit for labeling a nucleic acid polymer comprising: at least one nucleoside analogue that comprises a first reactive unsaturated group; and a reagent comprising a second reactive unsaturated group attached to a label.
 55. The kit of claim 54, wherein the first reactive unsaturated group comprises a 1,3-dipole, the second reactive unsaturated group comprises a dipolarophile and the first and second reactive unsaturated groups can react via [3+2] cycloaddition.
 56. The kit of claim 54, wherein the first reactive unsaturated group comprises a dipolarophile, the second reactive unsaturated group comprises α1,3-dipole, and the first and second reactive unsaturated groups can react via [3+2] cycloaddition.
 57. The kit of claim 55 or 56, wherein the 1,3-dipole comprises an azide group and the dipolarophile comprises an ethynyl group.
 58. The kit of claim 54, wherein the label is directly detectable.
 59. The kit of claim 58, wherein the label comprises a fluorescent agent.
 60. The kit of claim 54, wherein the label is indirectly detectable.
 61. The kit of claim 60, wherein the label comprises a hapten.
 62. The kit of claim 54 further comprising an aqueous medium and Cu(I).
 63. A kit for dually labeling a nucleic acid polymer, comprising at least one first nucleoside analogue that comprises a first reactive unsaturated group; at least one second nucleoside analogue that comprises a second reactive unsaturated group; a first reagent comprising a third reactive unsaturated group attached to a first label; and a second reagent comprising a fourth reactive unsaturated group attached to a second label.
 64. The kit of claim 63, wherein the first reactive unsaturated group comprises a first 1,3-dipole, the third reactive unsaturated group comprises a first dipolarophile, and the first and third reactive unsaturated groups can react via [3+2] cycloaddition; and wherein the second reactive unsaturated group comprises a second dipolarophile, the fourth reactive unsaturated group comprises a second 1,3-dipole, and the second and fourth reactive unsaturated groups can react via [3+2] cycloaddition.
 65. The kit of claim 63, wherein the first reactive unsaturated group comprises a first dipolarophile, the third reactive unsaturated group comprises a first 1,3-dipole, and the first and third reactive unsaturated groups can react via [3+2] cycloaddition; and wherein the second reactive unsaturated group comprises a second dipolarophile, the fourth reactive unsaturated group comprises a second 1,3-dipole, and the second and fourth reactive unsaturated groups can react via [3+2] cycloaddition.
 66. The kit of claim 64 or 65, wherein the first 1,3-dipole comprises a first azide group and the second 1,3-dipole comprises a second azide group or wherein the first dipolarophile comprises a first ethynyl group and the second dipolarophile comprises a second ethynyl group.
 67. The kit of claim 63, wherein the first and second label are directly detectable.
 68. The kit of claim 67, wherein the first label comprises a first fluorescent agent, the second label comprises a second fluorescent agent and the first and second fluorescent agent produce a dual fluorescence upon excitation.
 69. The kit of claim 63, wherein the first and second labels are indirectly detectable.
 70. The kit of claim 69, wherein the first label comprises a first hapten and the second label comprises a second hapten.
 71. The kit of claim 63 further comprising an aqueous medium and comprising Cu(I).
 72. A method of measuring cellular proliferation, comprising steps of: contacting a cell with an effective amount of a nucleoside analogue that comprises a first reactive unsaturated group such that the nucleoside analogue is incorporated into DNA of the cell; contacting the cell with a reagent comprising a second reactive unsaturated group attached to a label, such that a [3+2] cycloaddition occurs between the first and second reactive unsaturated groups; and determining an amount of label incorporated into the DNA to measure cellular proliferation.
 73. A method of measuring cellular proliferation in an organism, comprising steps of: administering to an organism an effective amount of a nucleoside analogue that comprises a first reactive unsaturated group such that the nucleoside analogue is incorporated into DNA of cells of the organism; contacting at least one cell of the organism with a reagent comprising a second reactive unsaturated group attached to a label, such that a [3+2] cycloaddition occurs between the first and second reactive unsaturated groups; and determining an amount of label incorporated into the DNA to measure cellular proliferation in the organism.
 74. The method of claim 72 or 73, wherein the first reactive unsaturated group comprises a 1,3-dipole and the second reactive unsaturated group comprises a dipolarophile or wherein the first reactive unsaturated group comprises a dipolarophile and the second reactive unsaturated group comprises a 1,3-dipole.
 75. The method of claim 74, wherein the 1,3-dipole comprises an azide group and the dipolarophile is an ethynyl group.
 76. The method of claim 72 or 73, wherein the label is directly detectable.
 77. The method of claim 76, wherein the label comprises a fluorescent agent.
 78. The method of claim 72 or 73, wherein the label is indirectly detectable.
 79. The method of claim 78, wherein the label comprises a hapten.
 80. The method of claim 72 or 73, wherein the step of contacting is performed in absence of Cu(I), and the reagent further comprises a Cu chelating moiety.
 81. The method of claim 72, wherein the cell is in a multi-well plate.
 82. A method for identifying an agent that perturbs cellular proliferation, comprising steps of: (a) contacting a cell with a test agent; (b) contacting the cell with an effective amount of a nucleoside analogue that comprises a first reactive unsaturated group such that the nucleoside analogue is incorporated into DNA of the cell; (c) contacting the cell with a reagent comprising a second reactive unsaturated group attached to a label, such that a [3+2] cycloaddition occurs between the first and second reactive unsaturated groups; (d) determining an amount of label incorporated into the DNA, wherein the amount of label indicates the extent of cellular proliferation; and (e) identifying the test agent as an agent that perturbs cellular proliferation if the amount of label measured in step (d) is less than or greater than the amount of label measured in a control application in which the cell is not contacted with the test agent.
 83. The method of claim 82, wherein step (b) is performed before step (a).
 84. The method of claim 82 further comprising the step of identifying the test agent as an agent that induces cellular proliferation if the amount of label measured in step (d) is greater than the amount of label measured in the control application.
 85. The method of claim 82 further comprising the step of identifying the test agent as an agent that inhibits cellular proliferation if the amount of label measured in step (d) is less than the amount of label measured in the control application.
 86. The method of claim 82, wherein the first reactive unsaturated group comprises a 1,3-dipole and the second reactive unsaturated group comprises a dipolarophile or wherein the first reactive unsaturated group comprises a dipolarophile and the second reactive unsaturated group comprises a 1,3-dipole.
 87. The method of claim 86, wherein the 1,3-dipole comprises an azide group and the dipolarophile is an ethynyl group.
 88. The method of claim 82, wherein the label is directly detectable.
 89. The method of claim 88, wherein the label comprises a fluorescent agent.
 90. The method of claim 82, wherein the label is indirectly detectable.
 91. The method of claim 90, wherein the label comprises a hapten.
 92. The method of claim 82, wherein the step of contacting is performed in absence of Cu(I) and the reagent further comprises a Cu chelating moiety.
 93. The method of claim 82, wherein the cell is in a multi-well plate.
 94. A method for identifying an agent that perturbs cellular proliferation in an organism, comprising steps of: (a) exposing an organism to a test agent; (b) administering to the organism an effective amount of a nucleoside analogue that comprises a first reactive unsaturated group such that the nucleoside analogue is incorporated into DNA of cells of the organism; (c) contacting at least one cell of the organism with a reagent comprising a second reactive unsaturated group attached to a label, such that a [3+2] cycloaddition occurs between the first and second reactive unsaturated groups; (d) determining an amount of label incorporated into the DNA, wherein the amount of label indicates the extent of cellular proliferation; and (e) identifying the test agent as an agent that perturbs cellular proliferation in the organism if the amount of label measured in step (d) is less than or greater than the amount of label measured in a control application in which the organism is not exposed to the candidate agent.
 95. The method of claim 94, wherein step (b) is performed before step (a).
 96. The method of claim 94 further comprising the step of identifying the test agent as an agent that induces cellular proliferation in the organism if the amount of label measured in step (d) is greater than the amount of label measured in the control application.
 97. The method of claim 94 further comprising the step of identifying the test agent as an agent that inhibits cellular proliferation in the organism if the amount of label measured in step (d) is less than the amount of label measured in the control application.
 98. The method of claim 94, wherein the first reactive unsaturated group comprises a 1,3-dipole and the second reactive unsaturated group comprises a dipolarophile or wherein the first reactive unsaturated group comprises a dipolarophile and the second reactive unsaturated group comprises a 1,3-dipole.
 99. The method of claim 98, wherein the 1,3-dipole comprises an azide group and the dipolarophile is an ethynyl group.
 100. The method of claim 94, wherein the label is directly detectable.
 101. The method of claim 100 wherein the label comprises a fluorescent agent.
 102. The method of claim 94, wherein the label is indirectly detectable.
 103. The method of claim 102, wherein the label comprises a hapten.
 104. The method of claim 94, wherein the step of contacting with a reagent is performed in vitro. 